Blood Sample Preparation Techniques and Protocols

Blood Sample
Preparation
Techniques and
Protocols for Your Lab
TA B L E O F CO N T E N TS
Introduction 3
Blood Sample Sources 3
Bone Marrow 3
Whole Blood 3
Apheresis Products 4
LRS Cones 4
Cord Blood 4
Buffy Coat 4
Overview of Methods for Blood Sample Preparation 5
Red Blood Cell (RBC) Depletion 5

RBC Lysis with Ammonium Chloride 5
Sedimentation 5
Immunomagnetic RBC Depletion 6
Buffy Coat Preparation 7
Peripheral Blood Mononuclear Cell (PBMC) Preparation from Whole Blood 7

PBMC Isolation Using Density Gradient Centrifugation 7
PBMC Isolation Using Immunomagnetic Cell Separation 9
Polymorphonuclear Cell (PMNC) Preparation from Whole Blood 10
What Is Immunomagnetic Cell Isolation? 11

Automated Immunomagnetic Cell Isolation 12
Sequential Isolation of Multiple Cell Types from a Single Sample 12
Cell Isolation from Large-Volume Samples 13
Isolation of Immune Cell Subsets from Whole Blood 14

Immunodensity Cell Separation Directly from Whole Blood 14
Immunomagnetic Cell Separation Directly from Whole Blood 15
Immunomagnetic Cell Isolation from PBMC or PMNC Fractions 15
Alternative Methods for Immune Cell Isolation 16
Cryopreservation of Isolated Cells 16
Protocols and Technical Tips 17
Blood Processing Protocols 18
Cell Isolation and Storage Protocols 24
Product Highlights 33
Introduction
Blood is an important source of immune and non-immune
cells that can either be used directly in biological assays
or further processed to isolate specific cell subsets. When
processing blood samples, carefully selecting protocols and Plasma
(~55%)
technologies can simplify blood processing, increase lab
efficiency, and reduce the risk of operator error. Together,
these effects can help to ensure accurate results in your
downstream assays.
Use this e-book to learn about different techniques and White blood cells
and platelets
protocols for processing blood samples, so you can choose (~4%)
the right method for your specific application. You may
also use it as a quick guide on techniques that may be Red blood cells
(~41%)
unfamiliar to you or for training new lab members.
Blood Sample Sources

The starting samples for processing blood and blood- Whole Blood
derived products can encompass a variety of sources,
Whole peripheral blood (i.e. circulating blood) is composed
including bone marrow, circulating whole blood, apheresis
of plasma, RBCs, leukocytes, and platelets. Leukocytes can
products, cord blood, and buffy coat. The blood or blood-
be further categorized into polymorphonuclear cells (PMNCs
derived sample source that you use will depend on what
or granulocytes) that contain granules (i.e. neutrophils,
tools are available to you for sample processing and your
eosinophils, and basophils) or mononuclear cells that have
intended downstream application. In this section, you will
a single nucleus (i.e. lymphocytes and monocytes).
learn about the different types of blood sample sources
and the applications for which they can be used.
Bone Marrow
Human bone marrow is a rich source of hematopoietic
stem and progenitor cells (HSPCs), which are responsible
for the production of blood cells such as leukocytes
(or white blood cells), red blood cells (RBCs or
erythrocytes), and thrombocytes (commonly referred
to as platelets).
WALLCHART
Frequencies of Human Cell Types in
Blood-Related Sources
www.stemcell.com/cells-wallchart
BLOOD SAMPLE PREPARATION 3

Apheresis Products
Apheresis is a process in which whole blood is drawn from
an individual, separated into components, and reinfused
back into the individual after a specific component (e.g.
plasma, platelets, or leukocytes) has been removed.
Leukapheresis is a form of apheresis in which leukocytes
are collected from a donor's circulating blood. Leukopaks
are enriched leukapheresis products that contain higher
Figure 1. LRS Cone
concentrations of leukocytes compared to whole blood
LRS Cone (Catalog #200-0093) containing primary human leukocytes.
or a buffy coat. They are an ideal starting source of
mononuclear cells and can be used for immune
Cord Blood
cell isolation.
Cord blood is collected from the placenta and umbilical
See page 18 for a protocol for processing leukopaks for cord immediately after birth. Given their enhanced
downstream cell isolation. capacity for progenitor cell proliferation, multilineage
differentiation, and self-renewal in vitro, cord blood-
Plateletpheresis is another form of apheresis in which
derived cells are an advantageous choice for developing
platelets are collected. This process may include a
screening assays for drug discovery, regenerative cell
leukoreduction step to remove leukocytes from the
therapy, and immune modulation. Compared to CD34+
platelet fraction. Leukoreduction creates leukocyte
hematopoietic stem and progenitor cells (HSPCs) derived
reduction system (LRS) cones as a byproduct, which can
from other tissues, HSPCs derived from cord blood are also
be used as a source of cells for downstream applications
more naïve, making them ideal for transplantation studies.
(see next section).
LRS Cones Buffy Coat

A buffy coat is a concentrated suspension of leukocytes
LRS cones, also known as LRS chambers, are used during
and is derived from whole blood or bone marrow following
the collection of leukapheresis products to reduce the
centrifugation. Generating a buffy coat from whole blood
leukocyte count in human blood collections (Figure 1).
samples can be used to concentrate large sample volumes
LRS cones contain a high concentration of leukocytes in
and reduce downstream cell separation handling.
a small volume, which can be further processed using cell
separation products to purify cell populations. See page 21 for a protocol for preparing a buffy coat from
For all of these blood sample sources, EasySep™ cell whole blood.
isolation kits can be used to isolate specific cell subsets for
future downstream analyses.
See page 20 for a protocol for processing LRS cones for

downstream cell isolation.
Start with the Right Cells

Start with a reliable supply of ethically sourced human primary
cells isolated from peripheral blood, mobilized peripheral
blood, cord blood, or bone marrow.
www.stemcell.com/PrimaryCells

Overview of Methods for Blood Sample Preparation
Red Blood Cell (RBC) Depletion
Contamination of your samples by red blood cells (RBCs) Standard ammonium chloride lysis may be too harsh for
can interfere with downstream analyses. Processing blood cord blood hematopoietic or immune progenitor cells.
samples for the isolation of specific cell types may therefore Therefore, alternative RBC depletion methods (e.g. RBC
require RBC depletion during the cell preparation step. sedimentation or immunomagnetic separation) may be
The following are examples of methods to deplete RBCs: better suited for such situations. Another factor to consider
is that cord blood can have very high platelet counts,
• RBC lysis with ammonium chloride
which may facilitate the formation of clots. It is therefore
• Sedimentation with dextran
important to use suitable anticoagulants (e.g. acid citrate
• Immunomagnetic cell separation with dextrose [ACD] or EDTA) and follow the manufacturer's
EasySep™ RBC Depletion Reagent cord blood-specific instructions when processing samples.
After RBC depletion is performed, the resulting sample Sedimentation

will contain only nucleated leukocytes that can be used
Sedimentation works on the basis that gravity will cause
for downstream assays or further processed to isolate a
larger and denser components to sediment faster than
specific cell subset.
materials that are smaller and less dense. Because of
their high rate of sedimentation, the largest and densest
RBC Lysis with Ammonium Chloride
components in a sample can be pelleted through an initial
A widely used method to remove RBCs from blood
low-force centrifugation. The supernatant can then be
samples is lysis with ammonium chloride. Homemade
re-spun, and through successive centrifugations,
or commercially available ammonium chloride solutions,
components with an increasingly lower rate of
also known as lysis buffers, lyse the RBCs in the blood
sedimentation can be isolated. Sedimentation is
sample while leaving the leukocytes intact. The remaining
inexpensive but generally results in lower purity than
leukocytes are then ready for downstream assays or further
other methods.
cell subset isolation. Although lysis buffer is inexpensive,
the process is difficult to automate. Dextran can be used for sedimentation by aggregating
RBCs. HetaSep™ is another alternative. These agents cause
See page 22 for a protocol for preparing a
the aggregated RBCs to settle much faster than dispersed
polymorphonuclear cell fraction using ammonium
cells, allowing for effective separation from nucleated cells
chloride lysis.
that remain in the supernatant.
Ammonium Chloride Solution from STEMCELL

Technologies is buffered and optimized for the gentle
lysis of RBCs while minimally affecting leukocytes. It can
be used for RBC lysis in preparations of human or mouse
peripheral blood, spleen, or bone marrow cells.

Immunomagnetic RBC Depletion
While RBC lysis with ammonium chloride and
sedimentation are widely used and cost-effective methods
to remove RBCs from blood samples, they can be harsh
on some blood sample sources, be difficult to automate,
or result in low purity. Alternatively, immunomagnetic
cell separation may be used to specifically deplete
RBCs from blood samples, leaving other cells behind.
Immunomagnetic cell separation involves targeting cells
for selection or depletion using antibodies or ligands
RBC Depletion Without Lysis, Washes,
directed against specific cell surface antigens. Labeled
or Centrifugation
cells are cross-linked to magnetic particles that can be
Use EasySep™ RBC Depletion Reagent to immunomagnetically
immobilized when an electromagnetic field is applied,
deplete RBCs and obtain highly purified and untouched
thereby separating them from unlabeled cells. For more leukocytes. The remaining leukocytes are ready for a variety of
information on immunomagnetic cell separation, downstream applications, including cell culture, RNA isolation,
see page 11. or enzyme activity testing.
www.stemcell.com/easysep-rbc-depletion
EasySep™ RBC Depletion Reagent immunomagnetically
targets RBCs for depletion to rapidly isolate untouched,
highly purified human leukocytes. This targeted RBC
depletion based on cell surface markers is more efficient
A Residual RBCs B Total Number of White
than lysis and results in fewer residual RBCs in the purified Blood Cells
80 1.5x107
sample (Figure 2). In addition, this method avoids exposing
Total Number of White Blood

% Residual Red Blood Cells
Cells Recovered per mL of

leukocytes to lysis buffer, which may be a concern for 60
1x107
Whole Blood
particular downstream assays and analyses. With an easy-
40
to-follow protocol, depleting RBCs with EasySep™ RBC
5x106
Depletion Reagent can take as little as 9 minutes when 20
performed manually. RBC depletion can also be automated 0 0

using RoboSep™ Cell Separation Instruments for increased eB
loo
d
dB
loo
d
fy B
loo
d
eM
arr
ow
Leu
kop
ak RBC
p™ on
niu
mo ide
m
hol Cor Buf ySe pleti nt Am Chlor
W Bon Eas De age
standardization and efficiency. Re
Ammonium EasySep™ RBC
Chloride Depletion Reagent
The EasySep™ RBC Depletion Reagent is compatible
with different blood-derived and blood-related products, Figure 2. EasySep™ RBC Depletion Reagent Provides Superior RBC
including: Depletion Compared to Ammonium Chloride Lysis
Different types of RBC-containing samples from normal healthy donors were
• Buffy coat processed to remove RBCs by using either ammonium chloride (NH4Cl) lysis
• Bone marrow or immunomagnetic depletion with EasySep™ RBC Depletion Reagent. (A)
The percentages of residual RBCs (Glycophorin A+/CD45-) in various samples
• Cord blood following the use of EasySep™ RBC Depletion Reagent were significantly lower
• Leukopaks than those in samples treated with ammonium chloride (mean ± SD; n = 31).
(B) RBC lysis with ammonium chloride and RBC removal using EasySep™ RBC
• Whole blood Depletion Reagent resulted in an equivalent total number of white blood cells
• LRS cones recovered from whole blood samples (mean ± SD; n = 37).
See page 9 for more information on RBC depletion using

EasySep™ Direct.
SAMPLE OFFER
Try Immunomagnetic RBC Depletion
in Your Lab
www.stemcell.com/sample-easysep

Buffy Coat Preparation
Low-speed centrifugation of whole blood or bone marrow Preparation of a PBMC fraction from whole blood is a
samples results in the density-based separation of plasma, common step prior to the isolation of specific immune
red blood cells (RBCs), and the leukocyte fraction. Also cell subsets. There are several methods for collecting and
known as a buffy coat, this leukocyte fraction can be used preparing PBMCs from human whole blood, including:
as the starting sample for further downstream isolation • Density gradient centrifugation
of specific cell populations using immunomagnetic cell • Immunomagnetic cell separation, e.g. with an
separation technologies. EasySep™ Direct Human PBMC Isolation Kit
PBMC Isolation Using Density Gradient

Centrifugation
Plasma
Plasma The most common PBMC isolation method is density
Whole
Whole Centrifugation
Centrifugation
Blood
Blood
Buffy Coat
Buffy Coat
gradient centrifugation, which relies on the varying
(White
(white bloodBlood
cells Cells
and
and Platelets)
platelets) densities of cells within a heterogeneous sample
RedBlood
Red Blood (Figure 3). First, the sample is layered over a density
Cells
Cells
gradient medium (e.g. Ficoll-Paque® or Lymphoprep™)
before being centrifuged. During centrifugation, each cell
See page 21 for a protocol for preparing a buffy coat type will sediment to its isopycnic point, which is the place
from whole blood. in the medium gradient where the density of the cells and
medium is equal.
Explore STEMCELL's selection of cell separation and
centrifugation tubes for blood sample processing. 1.077
Peripheral Blood Mononuclear Cell

Cell Number
(PBMC) Preparation from Whole Blood

Human peripheral blood mononuclear cells (PBMCs)
collected from blood samples are used in various research
applications, including flow cytometry, cell isolation,
cell culture, and cell-based assays. PBMCs are defined as
leukocytes with round nuclei and include lymphocytes
1.060 1.070 1.080 1.090 1.100
(i.e. T cells, B cells, and NK cells), monocytes, and
Density (g/mL)
dendritic cells.
Monocytes Neutrophils
Lymphocytes Eosinophils
Basophils RBCs
Figure 3. Densities and Relative Numbers of Human Blood Cells
To isolate mononuclear cells from whole blood, the density

of the density gradient medium should be 1.077 g/mL.
Lymphoprep™ and Ficoll-Paque® are similar media that
consist of saccharides and sodium diatrizoate. Both have a
density of 1.077 g/mL and are recommended for isolating
mononuclear cells from peripheral blood, cord blood, or
bone marrow using density gradient centrifugation.

To enrich for PBMCs using density gradient centrifugation,
whole blood is first diluted with phosphate buffered saline
(PBS) and then carefully layered over the density gradient
medium (e.g. Lymphoprep™). During centrifugation, the
cells with higher densities (i.e. granulocytes and RBCs)
sediment through the density gradient medium. The
PBMCs settle at the interface between the density gradient
medium and the plasma, from which they can be carefully
collected.
Cost-Effective Density Gradient Medium

Centrifugation
Plasma Isolate mononuclear cells from peripheral blood, cord blood,
Blood
PBMC or bone marrow by exploiting differences in cell density with
Density Gradient Lymphoprep™ density gradient medium.
Density Medium
Gradient Medium Granulocytes,
www.stemcell.com/lymphoprep
(Density: 1.077 g/mL) RBCs
See page 24 for a detailed protocol for isolating

mononuclear cells from whole blood by density gradient
centrifugation.
Although density gradient centrifugation is an inexpensive

cell separation technique, it has limited specificity, low
purity, low throughput, and cannot be automated.
Additionally, while it is a common laboratory technique,
density gradient centrifugation can be a slow and laborious
process that is difficult to master. Users need to carefully
layer their sample over the density gradient medium, Hassle-Free PBMC Isolation
centrifuge for 30 minutes with the brake off, and then Isolate PBMCs using density gradient centrifugation in as little
as 15 minutes with SepMate™, a specialized tube that allows
carefully harvest and wash the appropriate layer of cells.
users to quickly layer blood over the density gradient medium,
Consequently, isolating PBMCs from one sample typically prevents the layers from mixing, and facilitates easy harvesting
takes at least 45 minutes. of the target cells.
www.SepMate.com
Tip: Make the density gradient centrifugation step faster and

more efficient by using SepMate™ PBMC Isolation Tubes.
The special insert in SepMate™ tubes allows you to quickly layer
blood over the density gradient medium by preventing the layers
from mixing. This simplifies the density gradient centrifugation
protocol, resulting in fewer errors and reduced variability
among users.
VIDEO SAMPLE OFFER

How to Isolate PBMCs from Whole Blood Try SepMate™ in Your Own Lab
Using SepMate™ PBMC Isolation Tubes
www.stemcell.com/sample-sepmate
www.stemcell.com/sepmate-video

PBMC Isolation Using Immunomagnetic A Start: No Gate Isolated: No Gate
Cell Separation
Compared to time-consuming density gradient
Gly A PE
Gly A PE
centrifugation, immunomagnetic cell isolation can be used
to isolate PBMCs directly from blood in a single step that
can also be automated. For example, the EasySep™ Direct
Human PBMC Isolation Kit can be used to obtain PBMCs CD41 FITC CD41 FITC
in as little as 20 minutes by targeting RBCs, platelets, and Isolated: Density Gradient Isolated: EasySep™ Direct Human
B Centrifugation (No Gate) PBMC Isolation Kit (No Gate)
unwanted cells for immunomagnetic depletion (Figure 4).
Untouched PBMCs are simply poured into a new tube and
are immediately available for downstream applications such
as flow cytometry or cell culture. For additional information
SSC
on immunomagnetic cell separation, see page 11.
How does EasySep™ Direct PBMC isolation compare

FSC FSC
to density gradient centrifugation?
Figure 4. Typical EasySep™ Direct Human PBMC Isolation Profile
1. Reduces platelet contamination
(A) Starting with human whole blood from normal healthy donors, the typical
Density gradient centrifugation does not remove platelets, mononuclear cell content of the non-lysed final isolated fraction is 98.3 ± 2.8%
(gated on CD45). In the above example, the mononuclear cell contents of the
which can easily become activated and affect downstream
whole blood starting sample (lysed by ammonium chloride) and non-lysed final
applications. Removing platelets after the initial density isolated fraction are 27.0% and 98.6% (not gated on CD45), respectively.
(B) Mononuclear cells were isolated from whole blood samples using either
gradient centrifugation requires additional time-consuming
density gradient centrifugation or EasySep™ Direct Human PBMC Isolation Kit.
washing steps. The EasySep™ Direct Human PBMC Isolation Representative FSC vs. SSC flow cytometry plots (not gated on CD45).
Kit conveniently removes platelets during the cell isolation

step, so no additional washes are required.
Total Number Residual
A B Residual Cells C
of Cells Granulocytes
2. Reduces granulocyte contamination
% Residual Granulocytes
Recovered per mL of Blood
Granulocytes in blood samples degranulate and change

Total Number of Cells
% Residual Cells
density over time. This presents a challenge when using

density gradient centrifugation to process older blood
samples (> 48 hours after collection), as granulocyte
ets RBCs cytes
contamination of the PBMC fraction may occur. By Pla
tel
nu
lo
Timepoint (h)
Gra
specifically targeting unwanted cells for removal with Density Gradient EasySep™ Direct Human
Centrifugation PBMC Isolation
antibody complexes and magnetic particles, the EasySep™
Direct Human PBMC Isolation Kit results in less granulocyte Figure 5. EasySep™ Direct Human PBMC Isolation Kit Results in
contamination than density gradient centrifugation Fewer Contaminating Cells Than Density Gradient Centrifugation
(see Figure 5). PBMCs were isolated from whole blood samples using either density gradient
centrifugation or EasySep™ Direct Human PBMC Isolation Kit. Cells were
counted and analyzed by flow cytometry. (A) Density gradient centrifugation and
3. Enables automated PBMC isolation EasySep™ Direct Human PBMC Isolation Kit resulted in equivalent total numbers
PBMC isolation with EasySep™ Direct can be automated of nucleated cells recovered from 24-hour blood samples (mean ± SD; n = 14).
(B) Using EasySep™ Direct Human PBMC Isolation Kit to obtain PBMCs from
using the RoboSep™-S instrument to minimize sample 24-hour-old blood samples resulted in fewer residual platelets (CD41+), red
handling and free up valuable hands-on time. By automating blood cells (Glycophorin A+/CD45-), and granulocytes (CD66b+) than density
gradient centrifugation (mean ± SD; n = 15). (C) PBMC isolation from
all sample labeling and magnetic separation steps, you can 24-, 48-, 72-, and 96-hour-old blood samples using EasySep™ Direct Human
perform simultaneous cell isolations from up to four samples PBMC Isolation Kit resulted in fewer residual granulocytes than cell isolation
using density gradient centrifugation (mean ± SD; n = 3).
to increase sample throughput.

1 2 3 4 5 6
Figure 6. Protocol for EasySep™ Direct Human PBMC Isolation Kit
Polymorphonuclear Cell (PMNC)

Preparation from Whole Blood
Polymorphonuclear cells (PMNCs), also known as
granulocytes, are a collection of immune cell subsets with
enzyme-containing granules that can be released upon
cell activation. PMNCs include neutrophils, eosinophils,
basophils, and mast cells. To obtain a specific population
of PMNCs from whole blood, you can do any of the
following:
• Perform density gradient centrifugation followed by

ammonium chloride lysis of red blood cells (RBCs) in
the granulocyte fraction. See the protocol on page 22.
You can then isolate specific granulocyte populations
(i.e. neutrophils, basophils, or eosinophils) using VIDEO
immunomagnetic cell separation technologies. Watch How EasySep™ Direct Works
• Directly isolate granulocytes from whole blood, without www.stemcell.com/easysep-direct-video

lysis or density centrifugation, using immunomagnetic cell
separation techniques. For example, EasySep™ Direct kits
enable you to quickly and easily obtain pan-granulocytes
or a specific granulocyte subset (i.e. neutrophils, basophils,
or eosinophils). EasySep™ Direct uses immunomagnetic SAMPLE OFFER
cell isolation technology for separating PMNCs from whole Try EasySep™ Direct Human PBMC
Isolation Kit in Your Lab
blood (see page 9 for a comparison between density
gradient centrifugation and EasySep™ Direct). www.stemcell.com/sample-easysep

What Is Immunomagnetic
Cell Isolation?
Immunomagnetic cell separation uses magnetic particles Because of its speed and simplicity, immunomagnetic cell
to isolate target cells from heterogeneous cell suspensions. separation is commonly used to isolate highly purified
To accomplish this, the magnetic particles (also known populations of specific cell subsets. Immunomagnetic cell
as magnetic beads) are bound via antibodies to specific separation has several advantages, including:
cell surface proteins on the target cells. The sample is • High purity
then placed in an electromagnetic field that pulls on the
• Fast protocols
magnetic particles, bringing the labeled cells with them.
• Ease of use
The unlabeled cells remain in the supernatant, thus
physically separating target and non-target cells within • Low equipment costs
the sample. • High throughput
• Potential for automation
Immunomagnetic cell separation may be performed
• High cell viability
with or without columns. EasySep™ is a column-
free immunomagnetic cell separation technology that Both positive and negative selection can be performed using
eliminates the need for sample washing required by magnetic cell isolation methods (Figure 7). When positive
column-based methods, allowing you to get to your selection is performed, the supernatant can be discarded,
downstream applications without extended sample and the magnetically labeled cells of interest remain
manipulation. EasySep™ can be used on a variety of tissue immobilized until removed from the electromagnetic field.
types, including but not limited to whole blood, leukopaks, When negative selection is performed, the desired cells are
LRS cones, bone marrow, buffy coat, and cord blood. located in the supernatant.
Positive selection, negative selection, or cell depletion can
be performed with EasySep™ to obtain purified immune
cell subsets in as little as 8 minutes.
Figure 7. Comparison of Positive and Negative Selection
VIDEO E-BOOK
How EasySep™ Magnetic Cell Separation Cell Separation Techniques: Everything
Technology Works You Need to Know
www.stemcell.com/easysep-video www.stemcell.com/cell-separation-ebook

Automated Immunomagnetic Cell Isolation
Fully Automated, Walk-Away
Save hands-on time for laboratory scientists and
Cell Isolation
technicians performing immunomagnetic cell separation
RoboSep™ -S and RoboSep™ -16 instruments fully automate
procedures by adopting automated methods. The all cell labeling and separation steps of the EasySep™ procedure,
RoboSep™-S and RoboSep™-16 instruments are true minimizing sample handling and freeing up technician time.
walk-away, automated cell separation systems that retain www.RoboSep.com
the speed and simplicity of EasySep™ cell separation
technology and enable you to isolate cells from as many as
4 or 16 samples simultaneously. The column-free system
minimizes sample handling, eliminates the risk of cross-
100
contamination between samples, and reduces the risk of
exposure to dangerous pathogens. 90
80
% Purity
Sequential Isolation of Multiple Cell Types
from a Single Sample 70
Many analyses, such as chimerism testing, are often 60

performed on small-volume blood samples (e.g. pediatric
50
samples). In these situations, analysis of purified cell B Cells T Cells Myeloid NK Cells
subsets requires techniques that can efficiently isolate Cells
more than one cell type from an undivided starting Figure 8. Purity of Four Different Cell Types Isolated from 18 Different
sample. RoboSep™-16 can be used to sequentially isolate Samples Using RoboSep™-S Sequential Separation
Data kindly provided by Max Marschner, Supervisor, Florida Hospital
up to four different cell types from a single sample while
Tissue Typing Lab.
simultaneously processing up to four different samples
(Figure 8).
VIDEO TECH TIP

Automate Cell Isolation with the Isolate Multiple Cell Types from
RoboSep™-S Cell Separation Instrument a Single Sample
www.stemcell.com/robosep-s-video www.stemcell.com/sequential-isolation

Cell Isolation from Large-Volume Samples
Working with large-volume blood samples such as sample is placed in the magnet. Untouched cells can
whole blood or leukopaks can be time-consuming then be pipetted off into a new tube to be used for
and require splitting your sample to perform multiple downstream applications.
rounds of isolations. You can easily scale up your blood
See page 26 for more information on how to isolate
sample processing without splitting samples using the
multiple cell types from a single sample with
Easy 250 EasySep™ Magnet, which uses column-free
sequential isolation.
immunomagnetic EasySep™ cell isolation technology to
process up to a volume of 225 mL (or 12 billion cells) in a Need plasticware for your protocol? Explore our
single step (Figure 9). selection of flasks, tubes, pipettes, and more at
STEMCELL.com/cultureware.
With this technology, the labeled cells are pulled to the
sides of a standard T-75 cm2 cell culture flask when the
1 2 3 4
*Times are typical for next-generation negative selection kits. Time for each kit will vary depending on the exact isolation protocol used.
Figure 9. Typical Easy 250 EasySep™ Magnet Cell Isolation Protocol (Negative Selection)
See page 26 for a protocol on cell isolation with the Easy 250 EasySep™ Magnet.
DEMO OFFER VIDEO

See the Easy 250 EasySep™ How to Isolate Immune Cells from
Magnet in Action in Your Lab Large-Volume Samples Using the
Easy 250 EasySep™ Magnet
www.stemcell.com/demo-easy250
www.stemcell.com/easy-250-easysep-video

Isolation of Immune Cell Subsets
from Whole Blood
It is often necessary to isolate specific immune cell which unwanted cells are labeled and cross-linked to
subsets from blood for various applications, including RBCs. This results in the formation of complexes called
research, assay development, and diagnostics. Depending immunorosettes, which are much denser than the
on your needs and available equipment, you can either mononuclear cells being isolated. During centrifugation,
isolate specific immune cell subsets from whole blood the unwanted cells pellet with the RBCs, leaving the
or obtain peripheral blood mononuclear cells (PBMCs) desired cells in a layer above the density gradient medium.
or polymorphonuclear cells (PMNCs) first, followed by
Immunodensity cell separation does not require any
immune cell subset isolation. There are various approaches
specialized equipment beyond a centrifuge, can be
you can use to obtain specific cell subsets. The following
easily incorporated into established density gradient
methods will be covered in this section:
centrifugation protocols, and can be used to isolate
• Obtaining cell subsets directly from whole blood using specific cell subsets directly from whole blood. However,
immunodensity cell separation
the technique is limited to negative selection, relies
• Obtaining cell subsets directly from whole blood using on the operator’s blood sample layering technique,
immunomagnetic cell separation
and requires a high concentration of RBCs in the
• Isolating PBMCs or PMNCs first (see pages 7 and 10 for
starting sample.
more information), followed by immunomagnetic cell
separation to obtain specific cell subsets RosetteSep™ is a fast and easy immunodensity tool for
• Alternative methods for specific immune cell isolation from the isolation of untouched cells directly from whole blood.
PBMCs and PMNCs This approach significantly reduces sample handling time
and maximizes convenience by incorporating cell subset
Immunodensity Cell Separation Directly isolation into the centrifugation process (Figure 10).
from Whole Blood RosetteSep™ can be combined with SepMate™ PBMC
Immunodensity cell separation, also referred to as Isolation Tubes for even faster and easier immunodensity
erythrocyte rosetting, is a negative selection method cell separation.
that uses a combination of antibody-based labeling
See page 25 for a protocol for isolating specific cell subsets
and density gradient centrifugation. In this method, an
by immunodensity cell separation.
antibody cocktail is added to a whole blood sample in
1 2 3 4
Add RosetteSep™ Layer over density Centrifuge for 20 minutes* Collect cells
Antibody Cocktail gradient medium
Incubate
20 minutes
Plasma
Enriched cells
Whole Density gradient Density gradient medium
blood medium Highly purified cells
Red blood cells and
are left untouched
unwanted cells (rosetted)
*Use SepMate™ to reduce centrifugation time to 10 minutes with the brake ON.
Figure 10. Typical RosetteSep™ Cell Separation Protocol

Immunomagnetic Cell Separation Directly
from Whole Blood
Immunomagnetic cell separation is a technique that uses
antibody-linked magnetic particles to isolate desired cells
from heterogeneous mixtures. Because of its speed and
simplicity, immunomagnetic cell separation is one of the
methods most commonly used to isolate highly purified
populations of specific cell subsets. For more information
EasySep™ Direct is an immunomagnetic cell isolation Isolate Cells Directly from Whole Blood
method that can be used to isolate specific cell subsets Quickly obtain highly purified cells directly from whole blood
directly from whole blood. As described earlier for PBMC by immunomagnetically depleting RBCs and unwanted cells
in a single step without density gradient centrifugation,
and PMNC isolation from whole blood (see pages 7 and
sedimentation, or RBC lysis. Untouched cells isolated with
10), EasySep™ Direct may be used to immunomagnetically EasySep™ Direct are ideal for downstream applications such as
deplete RBCs and other unwanted cells in a single step gene expression analysis, functional assays, and flow cytometry.
without density gradient centrifugation, RBC lysis, or www.EasySepDirect.com
other pre-processing steps that can alter cellular function
or increase cell isolation time. EasySep™ Direct kits are
available for isolating a variety of specific cell types. Immunomagnetic Cell Isolation from PBMC
The isolated cells are untouched and highly purified, or PMNC Fractions
making them ideal for downstream applications like gene
After obtaining PBMCs or PMNCs from blood products
expression analysis, functional assays, or flow cytometry.
(see page 7 for how to isolate PBMCs and page 10 for
When using EasySep™ Direct, individual samples of how to isolate PMNCs), immunomagnetic cell isolation
0.5 - 30 mL can be processed in as little as 20 minutes. can be performed on either fraction to isolate target cells
To isolate cells from up to 16 samples simultaneously, from the heterogeneous PBMC or PMNC populations.
combine EasySep™ Direct with the EasyEights™ EasySep™ For example, EasySep™ is a versatile immunomagnetic
Magnet. For more information on how EasySep™ Direct cell separation technology that enables positive selection,
works, see page 9. negative selection, or cell depletion to obtain immune cell
subsets in as little as 8 minutes. Cell subsets isolated with
EasySep™ are highly purified, viable, functional, and ready
for a variety of downstream assays. For more information
SAMPLE OFFER
Try EasySep™ for Immune Cell
Subset Isolation
www.stemcell.com/sample-easysep
VIDEO
How to Obtain Purified Cells from Up to 16
Blood Samples at Once with EasySep Direct™
www.stemcell.com/easysep-direct-easyeights

Alternative Methods for Immune Cell Isolation
Several alternative methods exist for isolating immune
cell subsets from a PBMC or PMNC cell fraction obtained
from whole blood. One of the most common methods is
fluorescence-activated cell sorting (FACS), which uses flow
cytometry and fluorescent probes to sort heterogeneous
mixtures of cells. Fluorophore-tagged antibodies bind to
epitopes on specific antigens on the target cells within a
single-cell suspension. After tagging, the flow cytometer
focuses the cell suspension into a uniform stream of single
Cryopreserve and Store Viable Cells
cells, which is then passed through a set of lasers that
Use CryoStor® freezing media to mitigate temperature-induced
excite the cell-bound fluorophores to elicit fluorescent
molecular stress responses during freezing and thawing. This
emissions. Based on the emission wavelengths, the helps to maintain high viability and maximize cell recovery after
resulting photon signals are converted into a proportional long-term cryopreservation.
number of electronic pulses that assign a charge to the www.stemcell.com/cell-storage-media-overview.html
droplet that is formed around the cell. This charge enables
the droplet to either be deflected into collection tubes or
fall into the waste chamber. FACS has several advantages
over immunomagnetic cell separation, including the
Cryopreservation of
ability to: Isolated Cells
• Sort single cells Following blood sample processing, frozen vials of isolated
• Isolate cells based on intracellular markers (e.g. GFP) cells, such as peripheral blood mononuclear cells (PBMCs),
• Isolate cells based on surface marker expression levels can be stored for use in future assays (e.g. flow cytometry).
• Sort complex cell types with multiple markers at To do this, the cells are resuspended in cryopreservation
higher purity medium, cooled to extremely low temperatures using
a standard slow rate-controlled cooling protocol
Compared to FACS, immunomagnetic cell separation is a (approximately -1°C/minute), and then stored at liquid
much faster and simpler procedure, and is therefore often nitrogen temperatures (below -135°C) until required. When
the preferred method for isolating common cell types. needed, the cryopreserved immune cells can be thawed
for use in downstream applications. This process can be
There are also several less commonly used cell separation
standardized and automated with the portable ThawSTAR®
methods, such as buoyancy-activated cell sorting, aptamer-
CFT2 Automated Thawing System.
based cell isolation, and complement depletion. For more
information on these other methods, download our
See page 29 for a protocol for cryopreservation of PBMCs.
Cell Separation E-book.
See page 31 for a protocol for thawing frozen primary cells.
Ensure high cell viability and functional stability following

storage and thawing by using a reliable cryopreservation
medium, such as CryoStor ® freezing media.
Achieve reliable sample cryostorage with dependable,

high-quality cryovials and freezing containers, such as
Corning® CoolCell® LX (Catalog #200-0642).

Protocols and Technical Tips
This section provides optimized protocols and technical tips for processing blood samples, conducting downstream cell
isolations, and cryopreserving your cells of interest. Use these in-depth, comprehensive guidelines to consistently prepare your
starting samples and obtain your target cells.
For a complete list of cell separation protocols visit www.stemcell.com/cell-separation-methods
Blood Processing Protocols

How to Process a Leukopak for Downstream Cell Isolation 18
How to Process Leukocyte Reduction System (LRS) Cones/Chambers for Downstream Cell Isolation 20
How to Prepare a Buffy Coat from Whole Blood 21
How to Prepare a Polymorphonuclear Cell Fraction from Whole Blood Using Ammonium Chloride Lysis 22
How to Deplete Red Blood Cells (RBCs) Without Lysis Using EasySep™ Direct RBC Depletion Reagent 23
Cell Isolation and Storage Protocols

How to Isolate Mononuclear Cells from Whole Blood by Density Gradient Centrifugation 24
How to Isolate Specific Cell Subsets by Immunodensity Cell Separation 25
How to Isolate Multiple Cell Types from a Single Sample 26
How to Isolate Immune Cells from Large-Volume Samples Using the Easy 250 EasySep™ Magnet 26
How to Cryopreserve Peripheral Blood Mononuclear Cells (PBMCs) 29
How to Thaw Frozen Primary Cells 31

Blood Processing Protocols
How to Process a Leukopak for
Downstream Cell Isolation
The protocol below outlines how to process a fresh Protocol
or recently thawed leukopak for isolating cells using
manual or automated immunomagnetic and column-free BEFORE YOU BEGIN:
EasySep™ cell isolation kits. Following immunomagnetic cell Ensure that you handle the leukopak and its contents aseptically
within a biosafety cabinet.
separation, the untouched cells are immediately ready for
downstream analysis. Explore our wide range of EasySep™ Notes:
kits for isolating your cells of interest. For a frozen leukopak, follow the recommended thawing
protocol or use ThawSTAR® CB to reduce the risk of human
Materials error and standardize cell thawing.
• Leukopak (e.g. Catalog #70500)* For more tips and tricks, please view this webinar.
• Bag extractor stand

Part I: Transfer Leukopak Contents to a
• Transfer set spike
Sterile Container
• Transfer set tubing
1. To transfer the contents of the leukopak to a sterile
• Sterile container(s) container, place the leukopak in an extractor stand,
• 50 mL polypropylene conical tubes (e.g. Falcon® open the port on the bag, and insert the transfer
set spike.
Conical Tubes, 50 mL, Catalog #38010)
• Cell counting materials: 2. Close the plate of the extractor stand to apply
pressure to the bag.
- Hausser Scientific™ Bright-Line Hemocytometer
(Catalog #100-1181) 3. Open the valve and dispense the leukopak contents
- 3% Acetic Acid with Methylene Blue via the transfer set tubing into a sterile container.
(Catalog #07060) or Trypan Blue (Catalog #07050) Alternatively, if you do not have an extractor stand,
apply pressure to the bag or drain by gravity.
• Dulbecco's Phosphate Buffered Saline with 2% Fetal
Bovine Serum (PBS + 2% FBS, Catalog #07905) Part II: Prepare Leukopak Contents
• Recommended medium according to your cell isolation
kit's Product Information Sheet. Medium should be free BEFORE YOU BEGIN:
of Ca++ and Mg++. Recommended options include: It is recommended to perform an initial cell count of the
leukopak prior to any processing or washing steps. This number
- EasySep™ Buffer (Catalog #20144)
can then be compared to the cell count after the washing steps
- RoboSep™ Buffer (Catalog #20104) and used to calculate cell recovery.
- PBS + 2% FBS and 1 mM EDTA

Notes:
• Ammonium Chloride Solution
• If you are performing downstream cell isolation using an
(Catalog #07800; optional)
EasySep™ kit, consult the provided Product Information
• Cell isolation reagents and equipment: Sheet (PIS) to determine if red blood cell (RBC) lysis and
additional platelet removal steps are required. If RBC lysis
- EasySep™ cell isolation kit and additional platelet removal steps are NOT mentioned
(to obtain your cells of interest) or required, wash the leukopak contents as outlined in
Option 1. If the EasySep™ selection kit requires performing
- EasySep™ Magnet (compare magnets)
RBC lysis and platelet removal, refer to Option 2.
or RoboSep™ cell separation instrument
• Because of donor-to-donor variability, the content of RBCs
- Tube or flask for cell isolation and platelets in the leukopak may vary. After completing
the washing steps in Option 1, an additional RBC lysis step
*Certain products are only available in select territories.
and additional washes to remove platelets may be required
Please contact your STEMCELL Sales representative or the Product & Scientific
Support team at techsupport@stemcell.com for further information. (Option 2).

Option 1: Wash Leukopak Contents Part III: Count Cells and Prepare Cell Suspension
1. After the contents of the leukopak have been for Cell Separation
transferred to a sterile container, add an equivalent 1. Perform a cell count using either Trypan Blue or 3%
volume of buffer (PBS + 2% FBS recommended) to Acetic Acid with Methylene Blue. See the protocol
the container. on how to count cells with a hemocytometer for
2. Aliquot the diluted leukopak contents into cell counting instructions.
the appropriate tubes for centrifugation. We 2. Calculate the total volume required to resuspend
recommend using 50 mL conical tubes for the cells at the desired concentration, using the
centrifugation. following equation:
3. Centrifuge the cells at 300 x g for 10 minutes at
room temperature (15 - 25ºC) with the brake ON. Total Volume Required = Cell Concentration in Sample x Sample
Volume / Desired Final Cell Concentration
4. Carefully remove the supernatant without
disturbing the cell pellet.
Example:
5. Resuspend the cells by adding the recommended Desired final cell concentration = 5 x 107 cells/mL
medium to the sample. The cells are ready to in recommended medium
be counted. Cell concentration in sample = 1.825 x 107 cells/mL
6. If desired, pool all the cells from the different tubes Sample volume = 40 mL
into a single tube as follows: (1.825 x 107 cells/mL x 40 mL) / 5 x 107 cells/mL = 14.6 mL
The total volume required is 14.6 mL.
a. Resuspend the cells in a small volume of
the recommended medium.
3. After determining the volume required to obtain
b. Transfer the resuspended cells from individual
the desired cell concentration, centrifuge the cells
50 mL tubes into a single 50 mL tube. Make sure
at 300 x g for 10 minutes at room temperature
to rinse the tubes with the recommended
(15 - 25ºC) with the brake ON.
medium to recover as many cells as possible.
4. Remove the supernatant and resuspend the cells in
the desired medium with the appropriate volume
Option 2: RBC Lysis and Platelet Removal
to obtain the desired cell concentration.
1. After the contents of the leukopak have
been transferred to a sterile container, add
Note: For downstream manual isolation with EasySep™ or
Ammonium Chloride Solution to the sample at a
automated cell isolation with RoboSep™ instruments,
volume:volume ratio of 4:1. Mix by gently inverting resuspend the cells in EasySep™ Buffer, RoboSep™ Buffer, or
the tube. PBS + 2% FBS and 1 mM EDTA. The medium should be free of
Ca++ and Mg++.
2. Incubate on ice for 15 minutes.
3. Centrifuge the sample at 300 x g for 10 min at
room temperature (15 - 25ºC) with the brake ON.
4. Remove the supernatant and resuspend the cells.
5. Perform a washing step to remove platelets VIDEO
as follows: How to Process a Fresh or Frozen
Leukopak for Downstream Cell Isolation
a. Top up the tube with PBS + 2% FBS.
Centrifuge at 120 - 130 x g for 10 minutes at www.stemcell.com/leukopak-video
room temperature with the brake OFF.
b. Remove the supernatant. WEBINAR
c. Resuspend the cells in a small volume of the Leukopak Processing Tips & Tricks
recommended medium.
www.stemcell.com/leukopak-webinar
6. If additional platelet removal is required, perform
1 - 2 additional washing steps by repeating steps
5a through c

Part IV: Cell Isolation Using EasySep™ • 12 mL syringe
or RoboSep™ • Blunt-end needles, 16 Gauge (Catalog #28110)
Option 1: Manual Cell Isolation • 200 µL tips
1. Follow the instructions provided in the Product
• Serological pipettes (25 mL, e.g. Catalog #38005,
Information Sheet of the particular EasySep™ kit.
10mL, e.g. Catalog #38004)
For efficient isolation of immune cells from leukopaks, we

recommend using EasySep™ magnets that allow for large- BEFORE YOU BEGIN:
volume processing. Ensure that you handle the LRS cone and its contents aseptically
• Easy 50 EasySep™ Magnet (Catalog #18002) can process within a biosafety cabinet.
up to 40 mL and 4 x 109 cells.
• Easy 250 EasySep™ Magnet (Catalog #100-0821) can
Protocol
process up to 225 mL and 1.25 x 1010 cells.
• Compare across the different EasySep™ Magnets. 1. First, carefully clean the external surface of the LRS
cone and the tubing with 70% isopropyl alcohol
or ethanol.
Option 2: Automated Cell Isolation Using RoboSep™-S
2. Place the LRS cone, wide side down, over a
1. Add the cell suspension at the indicated cell 50 mL conical tube. This is the Collection Tube.
concentration in the EasySep™ Product Information
Sheet to a 14 mL (17 x 95 mm) polystyrene round- 3. With the LRS cone held over the Collection Tube,
bottom tube. use sterile scissors to cut the bottom tubing located
at the wider side of the cone, leaving approximately
2. Select the corresponding protocol on RoboSep™-S 2 to 3 cm of tubing.
and follow the onscreen prompts to load samples,
reagents, and tips. 4. Return the LRS cone to rest on the Collection Tube.
Then cut the top tubing located at the narrower
3. Press RUN to start the protocol. side of the cone, leaving approximately 1 cm of
4. After the run has been completed, unload the tubing. The sample will now start to drip into the
carousel. The isolated cells are ready for use. Collection Tube.
5. To facilitate the transfer of the sample into the
How to Process Leukocyte Reduction Collection Tube, an air flush can be performed
as follows:
System (LRS) Cones/Chambers for
a. Use a pipette to insert a sterile 200 µL tip into
Downstream Cell Isolation
the tubing at the top of the LRS cone.
The protocol below outlines how to process a leukocyte
b. Gently twist the tip several times to ensure
reduction system (LRS) cone for isolating cells using it is tightly positioned before releasing the
manual or automated immunomagnetic and column-free tip inside the tubing.
EasySep™ cell isolation kits. Following immunomagnetic cell c. Attach a blunt-end 16-gauge needle securely to
separation, the untouched cells are immediately ready for a 12 mL syringe, and use the syringe to push air
downstream analysis. into the 200 µL tip.
d. Continue to apply air until the sample, typically
Materials 8 to 10 mL, has been completely transferred
into the Collection Tube.
• LRS Cone (e.g. Catalog #200-0093)
6. Next, to rinse the cone, fill the syringe with a
• 50 mL tube rack (Catalog #200-0651)
buffer solution of PBS + 2% FBS. Gently dispense
• 50 mL polypropylene conical tubes the buffer solution through the 200 µL tip at
(e.g. Catalog #100-0090) approximately 2 mL/second. Be sure to move the
syringe in a circular motion to help wash the sample
from the sides of the cone.
Bovine Serum (PBS + 2% FBS, Catalog #07905)
7. Perform an air flush.
• Sterilized scissors in a paper heat-sealed sterilization pack

8. Repeat the wash and air flush steps (steps 6 How to Prepare a Buffy Coat from
and 7) 2 additional times until the cone
Whole Blood
has been rinsed with approximately 30 mL
of buffer solution. This protocol describes how to generate a buffy coat
from a whole blood sample, which can then be used
Notes: for downstream analyses or the isolation of specific
• Ensure that the collected wash solution does not contact cell populations using immunomagnetic cell isolation
the tubing at the bottom of the cone.
technologies such as EasySep™.
• After the final air flush, the sides of the cone should look
clear. The cone can then be discarded.
Materials
9. Top up the diluted sample to 50 mL with • Whole blood sample collected with anticoagulants
PBS + 2% FBS. • Recommended medium according to your cell
10. Securely cap the tube and gently mix the diluted isolation kit's Product Information Sheet.
sample by inverting the tube 5 to 6 times. Recommended options include:
11. Centrifuge the tube at 800 x g for 10 minutes with - EasySep™ Buffer (Catalog #20144)
the brake ON or with deceleration set to HIGH. - RoboSep™ Buffer (Catalog #20104)
- Dulbecco's Phosphate Buffered Saline with 2% Fetal
Note: Be careful not to disrupt the bottom layer containing the Bovine Serum (PBS + 2% FBS, Catalog #07905)
leukocytes and red blood cells.
• Tube for centrifugation
12. After centrifugation, use a 10 mL serological

Protocol
pipette to carefully remove the supernatant.
1. Add an equal volume of the recommended
13. Resuspend the sample (~10 mL) by gently flicking
medium to whole blood and mix gently.
or shaking the tube back and forth. The cells
are now ready for downstream applications but 2. Centrifuge at 800 x g for 10 minutes at room
may require additional processing steps for some temperature (15 - 25°C) with the brake OFF.
EasySep™ cell separation protocols. 3. Remove the concentrated leukocyte band (this
is the buffy coat) along with a small portion of
Note: If not used immediately, the cells can be stored at 2 - 8°C the plasma and concentrated red blood cells
for up to 48 hours. (RBCs). The target is to concentrate the leukocytes
approximately 5-fold while maintaining an
equivalent ratio of leukocytes to RBCs (e.g. collect
2 mL of buffy coat when starting with 10 mL of
whole blood).
VIDEO
How to Recover Cells from a Note: Although RosetteSep™ and most EasySep™ Direct kits
Leukocyte Reduction System (LRS) Cone have been optimized for use with whole peripheral blood, cells
can be enriched from buffy coat provided that:
www.stemcell.com/LRS-video
• RBCs are present at a ratio of at least 100 RBCs per
nucleated cell.
• The concentration of nucleated cells in the sample does not
exceed 5 x 107 cells/mL.
Explore various tools and products from STEMCELL

Technologies that have been optimized for the isolation
of cells from whole blood, buffy coat, the spleen, and
lymph nodes, and are not restricted to certain RBC ratios
or cell concentrations.

How to Prepare a Polymorphonuclear
Cell Fraction from Whole Blood Using
Ammonium Chloride Lysis
This protocol describes how to isolate polymorphonuclear Table 1. Recommended Volumes and Tube Sizes
cells from whole blood using density gradient centrifugation
Blood (mL) PBS + 2% FBS (mL) Lymphoprep™ (mL) Tube Size (mL)
followed by treatment with Ammonium Chloride Solution
for red blood cell (RBC) lysis. To learn more about cell 1 1 1.5 5
isolation from blood, see our Cell Separation E-Book.
2 2 3 14
Materials
3 3 3 14
• Whole blood containing anticoagulant*
4 4 4 14
• Centrifuge tube (e.g. Falcon® Round-Bottom Polystyrene
Tubes, 5 mL, Catalog #38007; Falcon® Round-Bottom 5 5 10 50
Tubes, 14 mL, Catalog #38008; or Falcon® Conical Tubes,
50 mL, Catalog #38010) 10 10 15 50
• Lymphoprep™ (Catalog #07801) 15 15 15 50

Bovine Serum (PBS + 2% FBS, Catalog #07905) 6. Add Ammonium Chloride Solution to the RBC
• Ammonium Chloride Solution (Catalog #07800)
pellet, completely filling the tube. Incubate on ice
for 10 minutes.
Please contact your STEMCELL Sales representative or the Product & Scientific 7. Centrifuge at 300 x g for 10 minutes at room
Support team at techsupport@stemcell.com for further information.
temperature with the brake set to LOW.
Protocol 8. Remove and discard the supernatant. Add PBS +

2% FBS to resuspend the pellet, then centrifuge at
1. Add Lymphoprep™ to a centrifuge tube. Refer to 120 x g for 10 minutes at room temperature with
Table 1 for the recommended volumes for various the brake OFF.
tube sizes.
9. Remove and discard the supernatant. Resuspend
2. Dilute whole blood with an equal amount of the resulting polymorphonuclear cells in
PBS + 2% FBS or other suitable culture medium. appropriate medium (e.g. PBS + 2% FBS).
3. Layer diluted blood on top of Lymphoprep™, Cell isolation kits are available for the positive selection,
being careful to minimize the mixing of blood
depletion, and negative isolation of total granulocytes and
with Lymphoprep™.
for the negative selection of neutrophils, eosinophils, and
4. Centrifuge at 800 x g for 20 minutes at room basophils from whole blood or the polymorphonuclear
temperature (15 - 25°C) with the brake OFF.
cell fraction.
5. Remove and discard the plasma layer, the band of
mononuclear cells, and Lymphoprep™, leaving the
RBC/granulocyte pellet intact.
Optional: At this point, the pellet may be transferred to a new

tube to avoid contamination by mononuclear cells remaining in
the tube.

How to Deplete Red Blood Cells (RBCs)
Without Lysis Using EasySep™ RBC
Depletion Reagent
In many laboratories, the standard protocols for obtaining STEMCELL Technologies has developed a simple protocol
leukocytes from human whole blood samples involve density (see below) that uses EasySep™ RBC Depletion Reagent to
gradient centrifugation or lysing red blood cells (RBCs) with immunomagnetically deplete RBCs without lysis, washing, or
ammonium chloride. However, these methods can be centrifugation steps. The resulting highly purified leukocytes
time-consuming, difficult to automate, and can leave are untouched and ready for downstream applications,
residual cell debris that may alter cellular function or including cell culture, RNA isolation, and enzyme activity
interfere with downstream assays. testing. EasySep™ RBC Depletion Reagent can also be used
on cord blood, bone marrow, buffy coats, or leukapheresis
products to meet all of your laboratory's needs.
Typical EasySep™ RBC Depletion Reagent Protocol
1 2 3 4 5 6
Add EDTA to sample and Add EasySep™ RBC Place sample into Pour or pipette off sample into Place the tube Pour or pipette off
dilute with PBS + 2% FBS. Depletion Reagent and magnet and incubate a new tube. Add EasySep™ into the magnet sample into a new
mix well. for 5 minutes*. RBC Depletion Reagent and and incubate for tube. Cells are now
mix well. 5 minutes*. ready for downstream
applications.
*Times will vary depending on the starting sample and EasySep™ magnet platform used.
To learn more, visit www.stemcell.com/RBCdepletion.
A B Residual RBCs C Total Number of White

Blood Cells
Total Number of White Blood Cells
200 80 1.5x10
Recovered per mL of Whole Blood
7
107
0.05 0.36
% Residual Red Blood Cells
10 6
149.7 60
Glycophorin A FITC
1x107
105
SSC (x10000)
99.5 40
10 4
5x10 6
103 49.2 20
-102 3.9 95.7

-1 0 0
-10 10
3 2
10 4
10 5
10 6
107 -103 101 103 10 4 105 10 6 107 d s
lo o od od r ro
w e si C niu
m
B Bl o Bl o Ma her ™ RB
mo id e
ole rd ffy ka p p n
A m C hlo r
CD45 APC CD45 APC
Wh Co Bu Bo
ne
Leu y S e e tio
Ea s D e pl g e nt
Rea
Ammonium EasySep™ RBC

Chloride Depletion Reagent
Figure 11. EasySep™ RBC Depletion Reagent Provides Superior RBC Depletion Compared to Ammonium Chloride Lysis
Different types of RBC-containing samples from normal healthy donors were processed to remove RBCs by using either ammonium chloride (NH4Cl) lysis or
immunomagnetic depletion with EasySep™ RBC Depletion Reagent. After RBC removal, the samples were stained with fluorochrome-conjugated anti-CD45 and
anti-Glycophorin A antibodies and analyzed by flow cytometry. Residual RBCs were identified as Glycophorin A+/CD45− events. (A) Typical flow cytometry plots
showing the residual RBCs in human whole blood samples following RBC removal with EasySep™ RBC Depletion Reagent (ungated). The typical percentage of
residual RBCs is 2 ± 3% (mean ± SD; n = 31). In the example above, the residual RBC content is 0.05%. (B) The percentages of residual RBCs in various samples
following the use of EasySep™ RBC Depletion Reagent were significantly lower compared to samples treated with ammonium chloride (mean ± SD; n = 31). (C) Both
RBC lysis with ammonium chloride and RBC removal using EasySep™ RBC Depletion Reagent resulted in an equivalent total number of white blood cells recovered from
whole blood samples (mean ± SD; n = 37).

Cell Isolation and Storage Protocols
How to Isolate Mononuclear Cells
from Whole Blood by Density
Gradient Centrifugation
This protocol describes how to isolate mononuclear cells, Table 2. Recommended Volumes and Tube Sizes
such as peripheral blood mononuclear cells (PBMCs),

Blood (mL) PBS + 2% FBS (mL) Lymphoprep™ (mL) Tube Size (mL)
from whole blood using density gradient centrifugation.
1 1 1.5 5
Materials
• Whole blood sample 2 2 3 14
• Density gradient medium (e.g. Lymphoprep™,

3 3 3 14
Catalog #07801)
4 4 4 14
• Dulbecco’s Phosphate Buffered Saline with 2% Fetal
Bovine Serum (PBS + 2% FBS, Catalog #07905) or other
5 5 10 50
suitable culture medium
• Centrifuge tube (e.g. Falcon® Round-Bottom 10 10 15 50
Polystyrene Tubes, 5 mL, Catalog #38007; Falcon®

15 15 15 50
Round-Bottom Tubes, 14 mL, Catalog #38008; or
Falcon® Conical Tubes, 50 mL, Catalog #38010)
5. Carefully harvest the cells by inserting the pipette
Protocol directly through the upper plasma layer to the
mononuclear cells at the interface. Alternatively,
BEFORE YOU BEGIN: remove the upper layer and then collect the cells.
Ensure all reagents are at room temperature (15 - 25°C).
6. Wash the harvested cells twice in the appropriate
buffer. The cells are now ready for downstream
1. Dilute the blood sample to a 1:1 volume ratio with applications.
the appropriate culture medium or PBS + 2% FBS.
2. Add a volume of density gradient medium to a

fresh tube according to the specifications of that
density gradient medium. If using Lymphoprep™,
see Table 2 for recommended volumes and
tube sizes.
3. Gently layer the diluted blood on top of the density

gradient medium. Take care not to mix the
2 layers. Speed Up Your PBMC Isolations
4. Centrifuge at 800 x g for 20 - 30 minutes with the Obtain PBMCs in just 15 minutes with SepMate™. The
brake OFF. specialized tube contains an insert that allows users to quickly
layer blood over the density gradient medium and prevents the
layers from mixing. Centrifugation is performed with the brake
ON, and the isolated PBMCs are simply poured off into a
fresh tube.
www.SepMate.com

How to Isolate Specific Cell Subsets
by Immunodensity Cell Separation
This protocol describes how to isolate cells from whole Table 3. Recommended Volumes and Tube Sizes for Density Gradient
Centrifugation Using Lymphoprep™
blood using RosetteSep™, an immunodensity cell
separation platform that isolates specific cell subsets
Sample Size Tube Size (mL) PBS + 2% FBS (mL) Density Gradient
during density gradient centrifugation. RosetteSep™ (mL) Medium (mL)
first cross-links unwanted cells to red blood cells (RBCs)

1 5 1 1.5
present in the sample to form immunorosettes. When the
sample is centrifuged over a density gradient medium, 2 14 2 3
the immunorosettes pellet, leaving highly purified cells

3 14 3 3
at the interface between the plasma and the density
gradient medium. This is a general procedure for using a 4 14 4 4
RosetteSep™ antibody cocktail to isolate a particular cell

5 50 5 15
type of interest; specific conditions may vary according to
the cell type being enriched. 10 50 10 15
Materials 15 50 15 15
• Whole blood sample

3. Centrifuge at 1200 x g for 20 minutes at room
• RosetteSep™ antibody cocktail for your desired temperature with the brake OFF.
cell subset Tip: Use SepMate™ to reduce centrifugation time to
10 minutes with the brake ON.
• Phosphate buffered saline containing 2% fetal bovine
serum (PBS + 2% FBS; e.g. Dulbecco's Phosphate 4. Remove the enriched cells from the density
Buffered Saline with 2% Fetal Bovine Serum, Catalog medium–plasma interface. Wash the enriched cells
#07905) with PBS + 2% FBS. Repeat.
• Density gradient medium (e.g. Lymphoprep™,
Catalog #07801) Note: It may be difficult to see cells at the interface, especially
when very rare cells are enriched. The following protocol
extension can be used to maximize recovery in these instances.
Protocol
BEFORE YOU BEGIN:

Optional Protocol Extension to Maximize
Ensure that the blood sample, PBS + 2% FBS, density gradient Recovery of Rare Cells
medium, and centrifuge are all at room temperature (15 - 25ºC).
a. Remove some of the density gradient medium along with
1. Add RosetteSep™ antibody cocktail to the sample the enriched cells.
and mix well. Incubate at room temperature
b. Add PBS + 2% FBS. Centrifuge at 300 x g for 10 minutes
(15 - 25°C) for 20 minutes.
with the brake on LOW. Discard supernatant. Repeat.
2. Dilute the sample with an equal volume of PBS + The enriched cells are now ready for use.
2% FBS and mix gently. Layer the diluted sample
on top of the density gradient medium using the
recommended volumes (Table 3). Be careful to
minimize mixing of the density gradient medium
and sample.

How to Isolate Multiple Cell Types
from a Single Sample
For situations in which sample size is limited and there is a Advantages of Sequential Separation
need to isolate multiple cell types, one may wish to perform • Allows you to work from a smaller starting volume
a sequential separation procedure. This avoids dividing • Maximizes cell recovery
the sample and ensures higher recovery of purified cell
• A quick and easy way to isolate multiple cell types
populations. Sequential separation is ideal for chimerism
• Can be automated using RoboSep™ to avoid
analysis, which is typically performed on low-volume blood cross-contamination
samples and requires techniques that can isolate more
than one cell type from a single starting sample for
How to Isolate Immune Cells from
lineage-specific analysis.
Large-Volume Samples Using the Easy 250
Most of our sequential separation protocols can isolate EasySep™ Magnet
two or more cell types from one sample as follows:
Manual cell isolations from large-volume samples, such as
1. The entire sample is labeled with an antibody leukopaks and whole blood samples, can be scaled up using
targeting the first cell type. These cells are then the Easy 250 EasySep™ Magnet with a standard
coupled to magnetic nanoparticles, and the sample
T-75 cm2 cell culture flask and EasySep™ reagents. This
is placed in the magnet. The supernatant with the
protocol describes the steps and key considerations when
unlabeled cells is removed and placed into a new
tube, leaving the first desired cell type in using the T-75 cm2 flask with the Easy 250 EasySep™
the magnet. Magnet. Please refer to Table 4 below for T-75 cm2 flask
2. The unlabeled fraction from the first separation compatibility with the Easy 250 EasySep™ Magnet as well
can then be labeled with an antibody targeting as key technical considerations when using this magnet for
the second cell type. These labeled cells are manual cell isolation.
isolated as described above, and the supernatant is
again removed and placed into a new tube for the Table 4. T-75 cm2 flask compatibility with the Easy 250
EasySep™ Magnet
final separation step.
3. The third cell type is isolated in the same manner, Compatibility Status Brand Supplier Catalog #
and the supernatant can simply be poured off.
353135 (plug-seal cap)/
STEMCELL #200-0500
This method can be applied to virtually any combination Compatible
Corning®
(officially recommended) 353136 (vented cap)/
of desired cell types. At STEMCELL Technologies, we have STEMCELL #200-0501
focused on optimizing various combinations specifically Compatible

Greiner Bio-One 658170
(suitable alternative)
for HLA labs, but the technique can be used in
130190
BioLife (Thermo)
many applications. 130193
229340
Incompatible CellTreat®
229341
General Tips for Sequential Separation (not recommended)
10062-862
VWR
Positive selection of a desired cell type must always be 10062-860
performed on an unlabeled cell population: (a) the original Corning® /Falcon 353110
starting sample, (b) the poured-off fraction from a positive Note: This table only includes the T-75 cm2 flasks for which compatibility has been
tested. If your flask type is not listed above, we suggest performing a pilot experiment
selection, or (c) the poured-off fraction or enriched cell
first to confirm the compatibility of that flask with our Easy 250 EasySep™ Magnet.
population from a negative selection. The flask should allow proper mixing of volumes up to 250 mL with a serological
pipette (e.g. Catalog #38005), and the flask neck should fit a 25 mL serological pipette
to allow mixing and volume collection.
We generally recommend isolating the rarest cell type first
(to minimize cell losses) and the most frequent cell type last,
although this convention cannot always be followed (please Custom protocols for specific applications are available
contact us for recommendations). by request. Please reach out to our Product and Scientific
Support team for more information.

Adjusting the Base Platform Height to Fit the Cell Isolation Cocktail, Magnetic Particles
T-75 cm2 Flask Type That Will Be Used (RapidSpheres™), and Buffer Addition
1. Place the Easy 250 EasySep™ Magnet on a flat 1. Add cocktail at the volume indicated in the Product
surface and ensure the spring-loaded side is pushed Information Sheet (PIS) of the kit being used.
in so that the T-75 cm2 flask can be easily inserted
a. Add the cocktail directly into the sample volume with
into the magnet.
a serological pipette and then gently pipette up and
2. If this is the first time this magnet is being used down 2 - 3 times.
with a particular type of flask, check if the base
b. Cap the flask. Agitate and rotate it several times to
platform height needs to be adjusted. To do so:
thoroughly mix.
a. Place the T-75 cm2 flask in the magnet to determine
how much the base platform height needs to be 2. Once the sample is mixed, incubate for the
raised or lowered, if necessary. Aim to have the recommended time provided in the PIS.
flask shoulder flush with the top of the magnet 3. Vortex the magnetic particles for at least 30 seconds
surface such that the flask cap and neck are not to ensure proper suspension.
hindered by the magnet.
4. Once the cocktail incubation is complete, add the
b. Once the correct height is determined, remove magnetic particles at the volume specified in the PIS
the flask. of the kit being used.
c. Place the magnet on its side and use a flathead a. Add the magnetic particles directly into the sample
screwdriver to adjust the height. volume with a serological pipette and gently pipette
d. To raise the flask base platform, use the up and down 2 - 3 times.
screwdriver to turn the drive bolt at the bottom of b. Cap the flask. Agitate and rotate it several times
the magnet counterclockwise. To lower the platform, to thoroughly mix.
turn the drive bolt clockwise. Take care not to
overtighten the drive bolt. 5. Once the sample is mixed, incubate for the
recommended time provided in the PIS.
e. Once the base height is adjusted, place the flask back
into the magnet to check if the shoulders of the 6. Following incubation, add the EasySep™ Buffer or
T-75 cm2 flask are flush with the magnet surface. recommended buffer at the volume indicated in the
PIS of the kit being used.
f. If needed, perform further adjustments to the
drive bolt. 7. Cap the flask. Agitate and rotate it several times to
thoroughly mix.
g. Once adjustments are complete, place the magnet in
the magnet stand to start the cell isolation protocol.
1 2 3 4
*Times are typical for next-

generation negative selection
kits. The time for each kit will
vary depending on the exact
isolation protocol used.
Figure 12. Isolating Immune Cells from Large-Volume Samples Using the Easy 250 EasySep™ Magnet

8. Once the sample is mixed, place the magnet on Resuspending Labeled Cells Within the Flask
the stand and push the spring-loaded side of the When using an EasySep™ positive selection kit, the PIS
magnet in so the flask can easily enter. will indicate that the positively selected cells need to be
9. Insert the flask into the magnet and push both resuspended and the magnetic separation step be repeated
buttons at the side so that the spring is released and the indicated number of times. To do so:
the flask sits tightly in the magnet.
1. Use a larger serological pipette (e.g. 25 mL) and add
10. Remove the cap and incubate the flask in the the indicated volume of EasySep™ buffer to the
magnet for the indicated amount of time in the PIS. flask. Alternate sides when dispensing the buffer
into the flask. Thoroughly mix the flask's contents
Collecting the Supernatant
between each addition of buffer to resuspend.
Once the separation time has elapsed, the supernatant can
2. To completely resuspend the cells from the edges of
be collected. the flask, use a smaller serological pipette to target
these cells for resuspension.
Note:
3. Cap the flask. Agitate and rotate it several times to
• When isolating cells with an EasySep™ negative isolation
thoroughly mix.
kit, the cells of interest will be present in the supernatant;
therefore, the supernatant should be saved. 4. Immediately place the flask into the Easy 250
• When isolating cells with an EasySep™ positive selection kit, EasySep™ Magnet.
the cells of interest will be attached to the sides of the flask
5. Push both buttons at the side of the magnet so that
while seated in the magnet; therefore, the supernatant can
be discarded.
the spring is released and the flask sits tightly in
the magnet.
1. To collect the supernatant, place a finger on the 6. Remove the cap and incubate the flask in the
flask shoulder to stabilize it. Use a 25 mL magnet for the amount of time indicated in the PIS.
serological pipette to aspirate the supernatant out of
the flask to maximize the volume that can be drawn. Note: These steps should be repeated the necessary number
2. When aspirating, position the pipette to draw from of times stated in the PIS.
the center of the flask and start aspirating from the

surface of the sample, moving down in a sweeping 7. Once complete, the cells can be resuspended in a
motion along the midline of the flask. Avoid desired volume (e.g. 50 mL), which can then be
touching the sides of the flask. transferred to a 50 mL tube to spin down for further
3. Once the bulk of the volume is removed, switch downstream applications.
to a smaller serological pipette to draw up the
residual volume.
4. Once the residual volume is aspirated out, cap the
T-75 cm2 flask and then push the spring side of the
magnet so that the original flask can be retrieved.
Figure 13. Using the Easy 250 EasySep™ Magnet Results in the
Isolation of Highly Purified Cells of Interest
Human immune cells were isolated from processed leukopaks using the
corresponding EasySep™ negative selection kits. Cell purity was measured
before isolation (Start) and after isolation with the Easy 250 EasySep™ Magnet
(Easy 250) and EasySep™ Magnet (Purple). Purity was assessed by staining
with cell surface markers for pan-T cells (CD3+), CD4+ T cells (CD3+CD4+),
CD8+ T cells (CD3+CD8+), or monocytes (CD14+CD45+) and analyzed by flow
cytometry (mean ± SD; n = 4 - 12).

How to Cryopreserve Peripheral Blood
Mononuclear Cells (PBMCs)
Cryopreservation is a commonly used method for the Materials
long-term storage of cells, including human peripheral blood • Cryopreservation medium (e.g. CryoStor® CS10,
mononuclear cells (PBMCs) isolated from whole blood or Catalog #07930; DMSO with FBS)
leukopaks. To cryopreserve PBMCs, the cells are resuspended
• Cryogenic vials (e.g. Catalog #38053)
in cryopreservation medium, cooled to extremely
low temperatures, and then stored at liquid nitrogen • Isopropanol freezing container (e.g. Nalgene® Mr. Frosty,
Sigma Aldrich Catalog #C1562) or a controlled-rate freezer
temperatures (below -135°C) until needed. When the cells
are required, the cryopreserved PBMCs can be thawed and • Isopropanol-free freezing container
then used in downstream applications, such as further cell (CoolCell® LX Corning® Catalog #200-0642)
subset isolation using EasySep™. • Pipettor (e.g. Corning® Lambda™ Plus Pipettor,
Catalog #38060)
A lab-made formulation of cryopreservation medium often
contains fetal bovine serum (FBS), a nutrient-rich ingredient • Pipette tips (e.g. Corning® Filtered Pipette Tips,
Catalog #38034)
that protects cells and helps them recover after thawing.
However, the use of FBS raises concerns about lot-to-lot
Protocol
variability and the risk of transmitting potentially infectious
Option 1: Cryopreservation with CryoStor® CS10
agents. FBS is usually not recommended for commercial or
clinical applications, such as the production of biologics. 1. Wipe down the outside of the CryoStor® CS10
container with 70% ethanol or isopropanol
Dimethyl sulfoxide (DMSO), another additive that helps
before opening.
protect cells against osmotic lysis, is also a widely used
2. Label cryogenic vials.
cryoprotectant in cryopreservation media.
3. Ensure the PBMCs are in a single-cell suspension.
Having an appropriate cryopreservation medium, cell Centrifuge the cells at 300 x g for 10 minutes to
concentration, and freezing rate ensures that PBMCs obtain a cell pellet.
maintain optimal viability and functionality upon thawing. 4. Carefully remove the supernatant with a pipette,
The following protocol outlines two options for successful leaving a small amount of medium to ensure the
cryopreservation of purified PBMCs: cell pellet is not disturbed.
• Using a serum-free cryopreservation medium: 5. Resuspend the cell pellet by gently flicking
CryoStor® CS10 (Catalog #07930), a serum- and animal the tube.
component-free cryopreservation medium containing 6. Add cold (2 - 8°C) CryoStor® CS10, mix
10% DMSO, provides a safe, protective environment thoroughly, and transfer the suspension to
for cells and tissues during the freezing, storage, and
a cryovial.
thawing processes.
• Using a serum-containing formulation: A lab-made Note: It is recommended to freeze isolated PBMCs at

formulation containing 10% DMSO with 90% FBS a concentration of 0.5 - 10 x 106 cells/mL. However, try
solution is a commonly used, inexpensive, and effective freezing the cells at multiple concentrations to determine
cryopreservation medium. which concentration gives the desired viability, recovery, and
functionality upon thawing. Higher cell concentrations for
freezing will need to be validated and optimized for specific
applications, as the major risk is the loss of viable cells.
7. Incubate cells at 2 - 8°C for 10 minutes.

8. Cryopreserve cells using a standard slow rate- 6. Mix the cells gently with 20% DMSO in FBS at
controlled cooling protocol (approximately -1°C/ a 1:1 volume ratio. The final cell suspension will
minute) in a controlled-rate freezer, an isopropanol be in 10% DMSO and 90% FBS. The final cell
freezing container (e.g. Nalgene® Mr. Frosty), or an concentration will be between 0.5 - 10 x 106
isopropanol-free freezing container (e.g. CoolCell® cells/mL. Rapidly transfer 1 mL of cell suspension
LX Corning®). If using an isopropanol freezing to each cryovial.
container, place the cryogenic vials into the
container and place it in a -80°C freezer overnight. Note: Try freezing the cells at multiple concentrations to
9. For long-term storage, transfer the vials of frozen determine which concentration gives the desired viability,
recovery, and functionality upon thawing.
PBMCs from the freezer or freezing container
to vapor-phase liquid nitrogen (below -135°C).
Minimize exposure to room temperature by 7. Place the cryogenic vials immediately into a
placing the vials on dry ice during transfer to freezing container. Place the container in a -80°C
liquid nitrogen. freezer overnight.
Note: Long-term storage at -80°C is not recommended. Note: Do not let the cells sit in cryopreservation medium at room
temperature. Keep on ice and transfer rapidly.
Option 2: Cryopreservation in 10% DMSO 8. For long-term storage, transfer the vials of frozen
with 90% FBS PBMCs to vapor-phase liquid nitrogen (below
-135°C). Minimize exposure to room temperature
BEFORE YOU BEGIN: by placing the vials on dry ice during transfer from
•
the -80°C freezer to liquid nitrogen.
Ensure all media are cold prior to starting this protocol.
• If there are safety concerns related to using FBS, a serum-
and animal component-free cryopreservation medium, such Note: Long-term storage at -80°C is not recommended.
as CryoStor® CS10, should be used.
1. Prepare 20% DMSO in FBS. Keep on ice.
Note: Do not put 100% DMSO on ice or it will form crystals.

Use a glass pipette for DMSO.
2. Label cryogenic vials.

3. Ensure the PBMCs are in a single-cell suspension.
Centrifuge the cells at 300 x g for 10 minutes to
obtain a cell pellet.
4. Carefully remove the supernatant with a pipette,
leaving a small amount of medium to ensure the
cell pellet is not disturbed.
5. Resuspend the PBMCs in cold FBS to a
concentration of 1 - 20 x 106 cells/mL. Keep on ice.

How to Thaw Frozen Primary Cells
Human primary cells are often cryopreserved (i.e. frozen) • DNase I Solution (1 mg/mL) (Catalog #07900)
for long-term storage in liquid nitrogen if they are not • Hemocytometer (e.g. Neubauer hemocytometer)
required immediately. Researchers with limited resources and cover slip
for processing fresh samples may also purchase frozen, • Trypan Blue (Catalog #07050)
ready-to-use primary cells, including mononuclear cells,
• Pipettor (e.g. Corning® Lambda™ Plus Pipettor,
purified immune cells, and hematopoietic stem cells. When
Catalog #38060)
thawing frozen cells, proper technique and handling ensure
• Pipette tips (e.g. Corning® Filtered Pipette Tips,
optimal viability, recovery, and functionality of the cells for
Catalog #38034)
downstream applications.
Protocol
This protocol describes how to thaw frozen primary cells.
As thawing protocols for specific cell types may vary, always Part I: Setup
refer to the recommended protocol received with your cells.
Note: It is recommended to thaw only 1 frozen cell vial at a time
Alternatively, automate this process and eliminate the to prevent prolonged exposure to DMSO at higher temperatures.
risk of contamination with the ThawSTAR® CFT2 and
ThawSTAR® CB Automated Thawing Systems, which 1. Warm the medium in a 37°C water bath. If
replace manual, water bath-based thawing. ThawSTAR® thawing cells for downstream cell separation,
systems not only eliminate the risk of contamination, but PBS with 2% FBS can be used.
deliver controlled thawing profiles. Utilize ThawSTAR® 2. When removing frozen cells from storage, it
CFT2 and ThawSTAR® CB to easily and consistently thaw is important to minimize exposure to room
temperature (15 - 25°C). If not proceeding directly
cryogenic vials and cryobags, respectively. To ensure
to thawing, place the cells on dry ice or in a liquid
maximum cell health by protecting your cells from transient
nitrogen container.
warming events, use the ThawSTAR® CFT2 Transporter
during vial transport. Part II: Thawing Cells
3. Wipe the outside of the cell vial with 70% ethanol
Materials or isopropanol.
• Human primary cells (frozen)

Note: It is important to work quickly in the following steps to
• Recommended medium. Options include: ensure high cell viability and recovery.
- Iscove's Modified Dulbecco's Medium (IMDM,
Catalog #36150) with 10% fetal bovine serum 4. In a biosafety cabinet, twist the cap a quarter-turn
(FBS) added
to relieve internal pressure and then retighten.
- DMEM with 4500 mg/L D-Glucose (Catalog #36250)
with 10% FBS added 5. Quickly thaw the cells in a 37°C water bath by
gently swirling the vial. Remove the vial when
- RPMI 1640 Medium (Catalog #36750) with
10% FBS added a small amount of ice remains. This should take
approximately 1 - 2 minutes. Do not vortex
- Phosphate buffered saline with 2% FBS
(e.g. Dulbecco's Phosphate Buffered Saline with the cells.
2% Fetal Bovine Serum, Catalog #07905)
Part III: Cell Washing and Counting
• 2 mL serological pipettes (e.g. Falcon® Serological
Pipettes, 2 mL, Catalog #38002) 6. Wipe the outside of the vial again with
70% ethanol or isopropanol.
• 25 mL serological pipettes (e.g. Falcon® Serological
Pipettes, 25 mL, Catalog #38005) 7. In a biosafety cabinet, measure the total volume of
the cell suspension using a 2 mL serological pipette.
• 50 mL conical tubes (e.g. Falcon® Conical Tubes, 50 mL, This value will be used in step 13 to calculate the
Catalog #38010) total number of cells provided. Place the cells back
into the vial to mix the suspension.

8. Remove a 20 µL aliquot of cells for counting.
If using Trypan Blue to assess viability:
for ≥ 1 x 106 cells, add a minimum of 20 µL of
medium and record the volume of medium added;
for < 1 x 106 cells, dilute directly in 20 µL of Trypan
Blue. Set the diluted aliquot aside until step 13.
For more details on performing cell counting with
a hemocytometer, please refer to the protocol on Thaw Your Frozen Cells Consistently
how to count cells with a hemocytometer.
Obtain reproducible thawing profiles with the portable
ThawSTAR® CFT2 Automated Thawing System. Cell vials are
Important: A viable cell count must be done on an aliquot thawed directly in the biosafety cabinet, eliminating the risk of
collected immediately after thawing (before washing). This will contamination and ensuring consistent thawing performance.
confirm the number of cells provided and track potential cell www.stemcell.com/products/thawstar-
loss in the wash process. Cell loss of up to 30% can be expected cft2-automated-thawing-system.html
during the washing steps.
9. Transfer the remaining cell suspension to a 50 mL 16. Gently add 15 - 20 mL of medium to the tube.
conical tube using a pipette.
17. Centrifuge the cell suspension at 300 x g for 10
10. Rinse the vial with 1 mL of medium and add it minutes at room temperature.
dropwise to the cells while gently swirling the
18. Carefully remove the supernatant with a pipette,
50 mL tube.
leaving a small amount of medium to ensure the
11. Wash by adding 15 - 20 mL of medium dropwise cell pellet is not disturbed. Resuspend the cell pellet
while gently swirling the tube. by gently flicking the tube.
12. Centrifuge the cell suspension at 300 x g for 10 19. The cells are now ready for use in downstream
minutes at room temperature (15 - 25°C). applications, such as cell culture with MethoCult™
13. If using Trypan Blue, perform a cell count on the or ImmunoCult™, and cell isolation with
diluted aliquot from step 8. EasySep™.
14. Carefully remove the supernatant (from step 12)

with a pipette, leaving a small amount of medium to
ensure the cell pellet is not disturbed. Resuspend the
cell pellet by gently flicking the tube.
15. If the cells are starting to clump, add 100 µg DNase I
Solution per milliliter of cell suspension and incubate
at room temperature for 15 minutes.
Note: Do not add DNase I Solution if the cells will be used for
DNA or RNA extraction.
VIDEO VIDEO
How to Thaw Frozen Human Primary Cells How to Quickly Thaw Frozen Cells with the
ThawSTAR® CFT2 Automated Thawing System
www.stemcell.com/thawing-protocol
www.stemcell.com/thawstarcft2-video

Product Highlights
The following is a curated list of products for blood sample preparation, cell isolation, and analysis. For a complete listing
of products to complement your research, visit www.stemcell.com.
Human Primary Cells* Cell Isolation and Depletion Products
Product Catalog # Product Catalog #
Human Peripheral Blood Leukopak, Frozen 200-0130 EasySep™ Direct Human T Cell Isolation Kit 19661
Human Peripheral Blood Leukopak, Fresh 70500 EasySep™ Direct Human B Cell Isolation Kit 19674
Leukocyte Reduction System (LRS) Cone 200-0093 EasySep™ Direct Human PBMC Isolation Kit 19654
70504 (ACDA) EasySep™ Direct Human Pan-Granulocyte Isolation Kit 19659

70501 (CP2D)
Human Whole Peripheral Blood EasySep™ RBC Depletion Reagent 18170
70508 (EDTA)
70507 (Na Heparin)
85415 (IVD)1
SepMate-15™
86415 (RUO)2
Human Peripheral Blood Mononuclear Cells, Frozen 70025
85450 (IVD)1
Human Peripheral Blood Mononuclear Cells, Fresh 200-0077 SepMate-50™
86450 (RUO)2

Cell Isolation Products for HLA Analysis
Density Media, Cryopreservation EasySep™ Direct HLA T Cell Isolation Kit 89671
Media, Buffers, and Other Reagents EasySep™ Serology Whole Blood CD3 Positive
Selection Kit
18981
EasySep™ Direct HLA B Cell Isolation Kit 89684

Product Catalog #
EasySep™ Serology Whole Blood CD19 Positive
18984
Lymphoprep™ 07801 Selection Kit
CryoStor® CS10 100-1061 EasySep™ Direct Human Total Lymphocyte Isolation Kit 19655
BloodStor® 55-5 07937 Cell Isolation Products for Cytogenetic Assays
Dulbecco's Phosphate Buffered Saline with

07905 EasySep™ Direct Human B-CLL Cell Isolation Kit 19664
2% Fetal Bovine Serum (PBS + 2% FBS)
Ammonium Chloride Solution 07800 EasySep™ Human CD138 Positive Selection Kit II 17877
RosetteSep™ Human Multiple Myeloma Cell

200-0642 15129
Corning® CoolCell ® LX Cell Freezing Container Enrichment Cocktail
1. SepMate™ (IVD) is available in Australia, Canada, Europe, the United Kingdom, and the
United States of America, where it is registered as an in vitro diagnostic (IVD) device for the
isolation of mononuclear cells from human whole blood, cord blood, and bone marrow
Antibodies by density gradient centrifugation. This product is also available in China, where it is
considered a non-medical device by the China Food and Drug Administration (CFDA),
and should therefore be used as general laboratory equipment.
Using the right antibody is an essential component for
2. SepMate™ (RUO) is available in regions where SepMate™ is not registered as an IVD
your research. Ensure that your downstream cell analysis, device and is for research use only (RUO).
including phenotyping and purity assessments, work

consistently by choosing from a line of high-quality
primary and secondary antibodies that are verified to work
Cell Isolation Instruments
with our cell isolation and cell culture reagents*.
Product Catalog #
Learn more at www.stemcell.com/antibodies.

RoboSep™-S 21000
*STEMCELL Technologies' antibodies are verified for use with STEMCELL's cell isolation RoboSep™-16 23000
products for select applications. Please consult the product information sheet for a complete
list of verified applications.

Copyright © 2023 by STEMCELL Technologies Inc. All rights reserved including graphics and images. STEMCELL Technologies & Design, STEMCELL Shield Design, Scientists Helping Scientists,
EasySep, HetaSep, RoboSep, RosetteSep, SepMate, EasyEights, RapidSpheres, ImmunoCult, STEMvision, and MethoCult are trademarks of STEMCELL Technologies Canada Inc. BloodStor, CryoStor,
and ThawSTAR are registered trademarks of BioLife Solutions, Inc. Nalgene is a registered trademark of Sigma-Aldrich. Lymphoprep is a trademark of Serumwerk Bernburg AG. The products sold
under the Lymphoprep brand name are also manufactured by Serumwerk Bernburg AG. Ficoll-Paque is a registered trademark of GE HealthCare Ltd. CoolCell, Corning, and Falcon are registered
trademarks of Corning Incorporated. All other trademarks are the property of their respective holders. While STEMCELL has made all reasonable efforts to ensure that the information provided by
STEMCELL and its suppliers is correct, it makes no warranties or representations as to the accuracy or completeness of such information.
PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED. FOR ADDITIONAL INFORMATION ON
QUALITY AT STEMCELL, REFER TO WWW.STEMCELL.COM/COMPLIANCE.

Blood Sample Preparation Techniques and Protocols

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Blood Sample Preparation Techniques and Protocols

Uploaded by

Copyright:

Available Formats

Blood Sample

Blood Sample Sources 3

Overview of Methods for Blood Sample Preparation 5

Red Blood Cell (RBC) Depletion 5

Buffy Coat Preparation 7

Peripheral Blood Mononuclear Cell (PBMC) Preparation from Whole Blood 7

Polymorphonuclear Cell (PMNC) Preparation from Whole Blood 10

What Is Immunomagnetic Cell Isolation? 11

Isolation of Immune Cell Subsets from Whole Blood 14

Cryopreservation of Isolated Cells 16

Protocols and Technical Tips 17

Blood Processing Protocols 18

Cell Isolation and Storage Protocols 24

Blood Sample Sources

BLOOD SAMPLE PREPARATION 3

LRS Cones Buffy Coat

See page 20 for a protocol for processing LRS cones for

Start with the Right Cells

BLOOD SAMPLE PREPARATION 4

After RBC depletion is performed, the resulting sample Sedimentation

Ammonium Chloride Solution from STEMCELL

BLOOD SAMPLE PREPARATION 5

Total Number of White Blood

Cells Recovered per mL of

performed manually. RBC depletion can also be automated 0 0

See page 9 for more information on RBC depletion using

BLOOD SAMPLE PREPARATION 6

PBMC Isolation Using Density Gradient

Peripheral Blood Mononuclear Cell

(PBMC) Preparation from Whole Blood

Figure 3. Densities and Relative Numbers of Human Blood Cells

To isolate mononuclear cells from whole blood, the density

BLOOD SAMPLE PREPARATION 7

Cost-Effective Density Gradient Medium

See page 24 for a detailed protocol for isolating

Although density gradient centrifugation is an inexpensive

Tip: Make the density gradient centrifugation step faster and

VIDEO SAMPLE OFFER

BLOOD SAMPLE PREPARATION 8

How does EasySep™ Direct PBMC isolation compare

Kit conveniently removes platelets during the cell isolation

Granulocytes in blood samples degranulate and change

density over time. This presents a challenge when using

BLOOD SAMPLE PREPARATION 9

Figure 6. Protocol for EasySep™ Direct Human PBMC Isolation Kit

Polymorphonuclear Cell (PMNC)

• Perform density gradient centrifugation followed by

• Directly isolate granulocytes from whole blood, without www.stemcell.com/easysep-direct-video

BLOOD SAMPLE PREPARATION 10

Figure 7. Comparison of Positive and Negative Selection

BLOOD SAMPLE PREPARATION 11

Many analyses, such as chimerism testing, are often 60

VIDEO TECH TIP

BLOOD SAMPLE PREPARATION 12

DEMO OFFER VIDEO

BLOOD SAMPLE PREPARATION 13

Figure 10. Typical RosetteSep™ Cell Separation Protocol

BLOOD SAMPLE PREPARATION 14

BLOOD SAMPLE PREPARATION 15

See page 31 for a protocol for thawing frozen primary cells.

Ensure high cell viability and functional stability following

Achieve reliable sample cryostorage with dependable,

BLOOD SAMPLE PREPARATION 16

For a complete list of cell separation protocols visit www.stemcell.com/cell-separation-methods