Cell preparation for genomics

Start smart to achieve new heights

in genomic analyses
 Improved sample
quality makes
the difference
We have a solution for every step in the preparation of
cells and nuclei for excellent genomic analysis results.
From tissue storage to quality control, our technology
will help your research achieve new heights.

Starting materials

FROZEN,
FRESH BLOOD
OR FFPE
TISSUE SAMPLES
TISSUE.

TISSUE TISSUE SAMPLE CELL

STORAGE DISSOCIATION CLEANUP ISOLATION

CELL
SORTING

FLOW
CYTOMETRIC
QUALITY
CONTROL

GENOMIC
ANALYSIS

2
Contents
4 Tissue storage, dissociation,
and cleanup

12 Magnetic cell isolation

18 Cell sorting and

flow cytometric quality control

22 Tips and information

Tissue storage,
dissociation, and cleanup
The preparation of viable single-cell suspensions or intact nuclei suspensions
is crucial when performing single-cell genomic analyses. Our sample preparation
products ensure efficient cell and nuclei preparation and offer flexibility
for fresh, frozen, or FFPE tissue processing.
 SAMPLE PREPARATION

Tissue storage
Short-term tissue storage helps you to gain flexibility Adm
Akr1b3
in sample processing. The MACS® Tissue Storage Aqp2
Aqp1
Aqp4
Arnt
Solution allows the optimal storage of fresh solid Atf6
Att4
4 Atf6b
tissues validated for up to 48 hours at 2–8 °C. It has Atg12
Atg7
Atg5
Atm
been tested with a variety of human and rodent Atr
Bid
Becn1

tissues including tumor, lung, spleen, brain, or skeletal Calr

Bnip31
Car9
Casp1
muscle. Storing tissues in MACS Tissue Storage Cd40jg
Ccl12
Cdkn1a
Cftr
Solution prevents the induction of necrosis and Chek2
Chek1
2 Crp
cellular stress while preserving the cellular Ddb2
Dnajc3
Ddit3
Edn1
composition and the activation status of cells. Epo
Fas
Ftb1
Gadd45a
Gadd45g
Gclc
Gclm
Grb2
Gsr
Gstp1
Hmox1
A Hsp90b1
Hsp90aa1
0 Hspa4
Hspa4j
100 Hspa5
Hus1
Itng
II1a
II1b
II6
Ldha
80 Mcl1
Mmp9
Mre11a
Nbn
Nfat5
Nqo1
Cell viability (%)

Parp1
Prdx1
60 -2 Pyr
Rad17
Rad51
Rad9a
Ripk1
Ripk3
Serpihe1
S1c2a1
40 S1c5a3
S1c9a3
Sqstm1
Tlr4
Tnf
Tnfrsf10b
Tnfrsf1a
Trp53
20 Txn1
Txnl4b
Txnrd1
-4 Vegfa
Ulk1
Xbp1
Xbc
Actb
0 B2m
Gapdh
fresh 24 h 48 h Gusb
Hsp90ab1
Time in storage 24 h 48 h
Time in storage
B
100 Figure 2: Expression of cellular stress-related genes in cells from
fresh and stored tumors. RNA was isolated from fresh and stored cells
from CT26 tumors and expression of cellular stress-related genes was
80
analyzed. The heat map shows the fold-change of expression rates
Cell frequencies (%)

compared to fresh tissue.

60

40

20

0
fresh 24 h 48 h
Time in storage

CD3+ CD3+CD25+ CD3+CD69+ CD3+CD4 +

CD3 CD8a
+ +
CD3 CD4
+ +
CD19 +
CD19+CD80+
CD25+ CD86+

Figure 1: Preserved cell viability and TIL composition of mouse

tumors stored in MACS Tissue Storage Solution. CT26 mouse
tumors were stored at 4 °C in MACS Tissue Storage Solution for 24 or
48 h. After dissociation, total cell viability (A) and TILs (tumor infiltrating
leukocytes) composition (B) were analyzed by flow cytometry. Cell
frequencies are referred to viable CD45+ cells. Additionally, frequencies
of CD3+ and CD19+ cell subsets refer to corresponding parental CD3+
and CD19+ populations respectively.

5
SAMPLE PREPARATION

Preparation of cells from solid tissues

The efficient dissociation of solid tissues into viable MACS® Tissue Dissociation Kits
single-cell suspensions is the most critical step in
We offer a wide variety of dissociation kits for human
the single-cell genomics workflow. The cellular
and rodent tissues. Our kits have lot-to-lot consistency
composition of the tissue must be preserved after
and contain highly-purified enzymes that ensure
dissociation, as well as cell surface epitopes to
standardized and reproducible dissociation results
further isolate specific target cells. Our gentleMACS™
with preserved cellular epitopes. For all dissociation
Technology enables the easy and fast dissociation
kits, we provide pre-defined protocols to ensure the
of solid tissues in a closed and sterile system that
maximal recovery of viable cells.
combines mechanical disruption and enzymatic
digestion to be as gentle to cells as possible.
Analysis of mouse tumor
gentleMACS Dissociators and Tubes heterogeneity at the single-cell level
The gentleMACS Octo Dissociator with Heaters allows Optimal dissociation of mouse tumors leads to the
for fully automated tissue dissociation. This dissociator clear identification of different cellular populations
has eight sample positions with integrated heating present in the tissue by single-cell gene expression
units to perform enzymatic incubation directly on analysis.
the instrument. Each position can run independently,
enabling you to process samples at any time, even if
other positions are being used. Over 40 pre-defined
programs have been developed along with our
tissue dissociation kits for optimal results. Moreover,
customized programs can be created to meet your
specific needs.

Luminal mammary cells Fibroblasts

B cells T cells
Plasma cells Basal mammary cells
Monocytes Dendritic cells

Figure 3: Single-cell gene expression analysis of mammary mouse

The gentleMACS Tube is an integral component
of gentleMACS Technology. Every element of the
tube has been engineered to ensure the highest
performance in the dissociation or homogenization
of tissue samples. Purple capped C Tubes are used
for tissue dissociation to get viable single-cell
suspensions from tissues, whereas orange capped
M Tubes are used for thorough tissue homogenization
for subsequent molecular analysis.
tumors. Tumors were dissociated using the Tumor Dissociation Kit,
mouse in combination with the gentleMACS Octo Dissociator with
Heaters. Single-cell RNAseq libraries were generated using the
Chromium™ Platform from 10x Genomics, and sequenced using
Illumina’s technology. Clustering was performed using Cell Ranger™
Software and visualized in Loupe Cell Browser, both from 10x Genomics.
Cell type classification was added to “graph-based” clusters based
on manual curation using known marker genes.

6
 SAMPLE PREPARATION

Preparation of nuclei from solid tissues

Some genomic applications require nuclei as starting
3.5
material. Also for certain tissues like snap-frozen
tissues, nuclei extraction is the only option for the 3.0

Extracted nuclei/mg (×10⁵)

analysis, since cells are heavily damaged during the 2.5
thawing process. For these applications we have
2.0
developed the Nuclei Extraction Buffer. Together with
the gentleMACS™ Octo Dissociators and C Tubes, the 1.5
Nuclei Extraction Buffer enables the fast extraction of 1.0
single nuclei at 4 °C within seven minutes from up to
0.5
eight samples in parallel. The Nuclei Extraction Buffer
has been successfully tested with a wide variety of 0
fresh and frozen tissues, including mouse brain, liver, 0 50 100 150 200 250
heart and kidney, mouse xenograft tumors, and OCT Tissue (mg)

embedded primary human tumors, such as melanoma, Liver Melanoma

breast, colon, and prostate. Heart Breast cancer
Kidney Colorectal cancer
Prostatic cancer

Figure 5: High yields of single nuclei after automated extraction

6.0 from frozen and OCT-embedded tissues. Single-nuclei suspensions
were obtained from snap-frozen mouse tissues, including liver, heart
and kidney and from OCT-embedded human tumors, including
Extracted nuclei/mg (×10⁵)

5.0
melanoma, breast cancer, colorectal cancer, and prostatic cancer
4.0 by using the Nuclei Extraction Buffer in combination with the
gentleMACS Octo Dissociator. Nuclei were stained with DAPI and
analyzed by flow cytometry using the MACSQuant Analyzer 10.
3.0

2.0

1.0

0
0 50 100 150 200 250 300
Tissue (mg)

Brain frozen 4T1 tumor frozen

B16 tumor frozen CT26 tumor frozen
Brain fresh 4T1 tumor fresh
B16 tumor fresh CT26 tumor fresh

Figure 4: High yields of single nuclei after automated extraction

from fresh and frozen tissues. Single-nuclei suspensions were
obtained from fresh and snap-frozen mouse brain and syngeneic mouse
tumors using the Nuclei Extraction Buffer and gentleMACS Octo
Dissociators. Nuclei were stained with DAPI and analyzed by flow
cytometry using the MACSQuant® Analyzer 10.

VISIT
Visit our website for more
information on nuclei extraction: Figure 6: Single-nuclei suspension from mouse liver. Single nuclei
 iltenyibiotec.com/
m were extracted from snap-frozen mouse liver using the Nuclei Extraction
Buffer in combination with the gentleMACS Octo Dissociator. Immediately
nucleiextraction after nuclei extraction, nuclei were stained using DRAQ5™ Staining
Solution. The image shows an overlay of DRAQ5™ (pink) and brightfield.

7
SAMPLE PREPARATION

Sample cleanup
Cell suspensions often contain unwanted material, The Debris Removal Solution is a ready-to-use
such as cell aggregates, dead cells, debris, and red density-gradient reagent that allows the fast and
blood cells, which can interfere with proper cell convenient removal of debris in cell suspensions
count and the results of your genomic downstream containing fragile cells, including cells from adult
application. When performing single-cell mouse brain, heart, liver, and kidney. Both dead cell
transcriptomic analysis, only viable cells will or debris removal, in combination with red blood cell
generate reliable genomic information. lysis, provide the perfect solution to increase the yield
of viable target cells for downstream applications.
Our MACS® SmartStrainers and removal reagents
efficiently clean and prepare your samples for
genomic analysis.
Before dead cell After dead cell
removal removal
With their special design, MACS SmartStrainers enable
filtration without clogging and easily fit onto 15 mL and
50 mL tubes for the fast removal of larger particles and
cell aggregates in cell suspensions or blood samples.
PI

38% 90%

Forward scatter

Figure 7: Efficient removal of dead cells even from samples with

high dead cell content. The Dead Cell Removal Kit was used to
remove dead cells from PBMC samples. Dead cells are defined as
PI-positive cells and viable cells as PI-negative.

A Before sample cleaning

1000 1000
Debris Erythrocytes
Side scatter

750 750
Anti-Ter-119

500 500
89.8%
250 250

0
9.4% 0 10.2%
0 250 500 750 1000 0 250 500 750 1000

Forward scatter
Sequence viable nucleated cells B After sample cleaning
and ensure proper cell count 1000 1000

Dead cells, red blood cells, and debris can hamper 750 750
Side scatter

Anti-Ter-119

your genomic analysis and should therefore be 500 500

removed from the sample. 3.2%

250 250

73.9% 96.8%
Use the Dead Cell Removal Kit for the fast and 0
0 250 500 750 1000
0
0 250 500 750 1000
straightforward magnetic depletion of dead cells Forward scatter
from peripheral blood mononuclear cells (PBMCs),
Figure 8: Enrichment of highly viable neural cells from adult
cryopreserved cells, or cell suspensions from solid mouse brain. (A) The cell suspension from adult brain contains a
tissues containing robust cells, such as epithelial cells, significant amount of cell debris and erythrocytes. (B) Viable neural cells
are enriched after efficient removal of debris and erythrocytes using
tumor cells, and immune cells. the Debris Removal Solution and Red Blood Cell Lysis Solution (10×).

8
 SAMPLE PREPARATION

Cleanup of tumor samples improves the Single-cell gene expression

quality of single-cell genomic analysis
Effective filtration and removal of dead cells and red
blood cells prior to single-cell sequencing of mouse
tumors helps to increase the cell recovery rate, leading
to higher sequencing data quality.
analysis of adult mouse brain
Optimized dissociation of adult mouse brain, including
debris removal and red blood cell lysis, allows for
the efficient single-cell sequencing analysis of major
neural cell types from adult mouse brains.

5,000 Oligodendrocytes Macrophages,

Cldn11 monocytes, DCs
Number of cells recovered

5 Pf4, Plac8, Cd209a

4,000 10 Vascular cells
15 ImmN 12 B cells Clnd5
Sox11 20 lgkc
3,000 19
14 T cells 17 2
5 mNEUR Cd3g 22
2,000 Syt1 OEG
8 VMLC 23 VSMC
Npy Slc6a13 13 Acta2
18
UMAP_2

0 Pericytes 11
1,000 9
0 21 3 Kcnj8
7
0 –5 Choroid plexus cells
4 1 Ttr
A B C D A B C D A B C D
6
Cleanup step Microglia Astrocytes
–10 Tmem119 Gja1
Figure 9: Impact of sample cleanup in single-cell RNA sequencing Arachnoid barrier cells
of three mouse colon tumor replicates. Tumors were dissociated with Ependymal cells Slc47a1 16
Ccdc153 10
gentleMACS™ Octo Dissociator with Heaters and Tumor Dissociation –15
Kit, mouse followed by filtration with a 70 μm MACS® SmartStrainer (A);
–10 –5 0 5 10
red blood cells were removed with Red Blood Cell Lysis Solution (10×) (B);
and dead cells were removed once (C) or twice (D) using the Dead Cell UMAP_1
Removal Kit. Aliquots were taken at each step and single-cell RNAseq
libraries were generated with the Chromium™ Platform from 10x 0 6 12 18 ImmN: Immature neurons
Genomics and sequenced using Illumina’s technology. Data was 1 7 13 19 mNEUR: Mature neurons
analyzed using Cell Ranger™ software from 10x Genomics. 2 8 14 20 OEG: Olfactory ensheathing glia
3 9 15 21 VMLC: Vascular and
4 10 16 22 leptomeningeal cells
5 11 17 23 VSMC: Vascular smooth
muscle cells

Figure 10: Single-cell gene expression analysis of adult neural cells.

Whole adult mouse brains were dissociated using the Adult Brain
Dissociation Kit, mouse and rat in combination with the gentleMACS
Octo Dissociator with Heaters. The kit includes the reagents to perform
debris and erythrocyte removal. Afterwards, cells were applied to
the Chromium™ Platform from 10x Genomics and single-cell libraries
were sequenced using Illumina’s technology. The Uniform Manifold
Approximation and Projection (UMAP) was used to visually represent
the clusters of the different neural populations based on their gene
expression profile.

9
Magnetic cell isolation
The genomic analysis of PBMCs or specific cell types from a sample can be time
consuming and expensive, especially when target cells are present at low frequencies.
Cell isolation helps you to increase the sensitivity of the analysis by excluding
the background signals from non-target cells, while saving time and sequencing costs.

Select the best isolation approach
MACS® Technology enables the magnetic separation
of cell populations by targeting surface antigens with
specific antibodies conjugated to superparamagnetic
beads. Labeled cells are magnetically retained in a
separation column, from which they can be eluted.
The fast and gentle isolation minimizes cellular
changes and ensures high viability.
Based on MACS Technology, we offer different cell
separation solutions to fit your every need.
1. Magnetic labeling
Cells of interest are
magnetically labeled with
MACS MicroBeads.
2. Magnetic separation
Cells are separated in a
MACS Column placed in
a MACS Separator.
The flow-through fraction
can be collected as the
negative fraction depleted
of the labeled cells.
3. Elution of labeled cells
The column is removed from
the separator. The retained
cells are eluted as the enriched,
positively selected cell fraction.
Figure 11: Three easy steps of MACS MicroBead Technology to isolate
your cells of interest.
CELL SEPARATION
Separation solutions to fit every need
StraightFrom® MicroBeads
• Direct isolation of leukocyte subsets from
blood samples in less than 30 min without
density gradient centrifugation or red
blood cell lysis
• Avoid cellular changes through
density gradient centrifugation
and red blood cell lysis
MACS MicroBeads
• Gentle, positive isolation ensures
highest purity, even for rare cells
• Applicable to virtually any cell type,
and tissue, from PBMCs to complex tissues
• Beads do not need to be removed
for downstream applications
MACS Isolation kits
• Ideal method to isolate PBMCs and cells
without a defined lineage-specific marker,
such as tumor cells or neurons
• Target cells are label free and can be used
for subsequent magnetic isolations
VISIT
Visit our website for more
information on MACS Technology:
 iltenyibiotec.com/macstech_
m
genomics
11
CELL SEPARATION

Automation minimizes Direct immunomagnetic isolation

user variation and errors
Whether you work with few samples or in a high-
throughput setting, automation helps to streamline
your cell separation. Our instruments for cell
separation allow different levels of automation
for scalable sample throughput, while standardizing
cell separation processes.
of PBMCs from whole blood
prevents cellular stress
Gentle and automated immunomagnetic isolation
of PBMCs directly from whole blood prevents
activation of cellular stress responses in comparison
to density gradient centrifugation methods.

For high reproducibility, the autoMACS® Pro Separator

Ficoll® and
is the ideal instrument because it performs fully Value RBC lysis Ficoll®
automated magnetic cell separation while minimizing Akr1b3
Adm
Aqp1
temperature changes and user variation. Besides -3 Aqp2
Aqp4
Arnt
magnetic cell separations of particular target cells, Atf6
Att4
Atf6b
Atg12
isolation of PBMCs from blood samples and removal 0 Atg7
Atg5
Atm
of dead cells using the Dead Cell Removal Kit are also Atr
Bid
Becn1
Bnip31
possible with the autoMACS Pro. 3
Calr
Casp1
Car9
Ccl12
Cd40jg
Cdkn1a
Cftr
Chek1
Chek2
Crp
Ddb2
Ddit3
Dnajc3
Edn1
Epo
Fas
Ftb1
Gadd45a
Gadd45g
Gclc
Gclm
Grb2
Gsr
Gstp1
Hmox1
Hsp90aa1
Hsp90b1
Hspa4
Hspa4j
Hspa5
Hus1
Itng
II1a
II1b
II6
Ldha
Mcl1
Mmp9
Mre11a
Nbn
Nfat5
Nqo1
Parp1
Prdx1
Pyr
Rad17
Rad51
Rad9a
Ripk1
Ripk3
Serpihe1
S1c2a1
S1c5a3
S1c9a3
Sqstm1
Tlr4
Tnf
Tnfrsf10b
Tnfrsf1a
Trp53
Txn1
Txnl4b
Txnrd1
Ulk1
Vegfa
Xbp1
Xbc
Actb
B2m
Gapdh
Gusb
Hsp90ab1

Figure 12: Immunomagnetic-based isolation of PBMCs from whole

blood prevents cellular stress. PBMCs were prepared using density
gradient centrifugation (Ficoll®) or using the Whole Blood PBMC
Isolation Kit, human and the Sedimentation Kit 2. Red blood cells were
removed using the Red Blood Cell Lysis Solution (10×) from an aliquot
of the PBMCs prepared using Ficoll® (Ficoll® and RBC lysis). RNA was
isolated and expression of cellular stress-related genes was analyzed.
The heat map shows the fold-change of gene expression rates
compared to PBMCs prepared with immunomagnetic beads.

12
 CELL SEPARATION

Isolation of TILs significantly increases Tumor cell isolation allows sensitive

the sensitivity of single-cell immune and robust detection of somatic
profiling analysis mutations via NGS
Magnetic isolation of TILs after tumor dissociation Isolation of tumor cells enables accurate detection
improves the sensitivity of single-cell RNA sequencing of single nucleotide polymorphisms (SNP)
of T and B cell receptors, highlighting clonotypes in human solid tumors improving detectability
otherwise poorly or unrepresented in the bulk sample. of loss of heterozygosity (LOH).

A Frequency ref pos TCRb CHECK2 CTNNB1

30 c.349A>G p.R117G c.133T>C p.S45P
28
26
Isolated
100 100
of cells

Variant frequency (%)

Variant frequency (%)

24
Bulk
22
20 80 80
18
# of cells

16
60 60
Number

14
12
10
8 40 40
6
4
2 20 20
0
CDR3s
CDR3 clonotypes 0 0
Thymoma Thymoma
B Frequency ref pos BCR IGH

120 Bulk tumor Isolated tumor cells

Frequency ref bulk TCRb
100 Isolated
# of cellsof cells

30
80
28
60
Bulk Figure 14: Tumor cell isolation enables an accurate detection
26 Bulk
40
24 Isolated of SNP zygosity in solid tumors. Tumor cells were isolated from
20
22
dissociated human thymoma tumors. DNA from bulk tumor or isolated
Number

20
10
18 tumor cells was used to produce exome-capture sequencing libraries
# of cells

8
16
6
14
with Illumina’s technology. The variants CHECK2 c.349A>G p.R117G
12
4 and CTNNB1 c.133T>C p.S45P are depicted. While the frequency of the
10
2
8 indicated mutation in the CHECK2 gene in the bulk tumor suggested
06
IGH CDR3s heterozygous conditions, in the isolated tumor cell fraction LOH events
4
BCR IgH CDR3 clonotypes
2 could be detected. Furthermore, the somatic mutation c.133T>C p.S45P
0
CDR3s in the CTNNB1 gene could only be detected in isolated tumor cells.
Bulk Isolated

Figure 13: Isolation of TILs improves the sensitivity of single-cell

immune profiling. After dissociation and cleanup of human ovarian
carcinoma samples, T cells and B cells were isolated using MACS
Technology. Single-cell immunoprofiling was performed on bulk
Frequency ref Ori BCR IGH
samples or isolaed T and B cells using 10x Genomics’ and Illumia’s
technologies.
120
100
The graphs show the top 50 TCR (A) and topBulk25 BCR
(B) CDR3
80 clonotypes ranked by their abundancy in the isolated T or B
Isolated
cell fraction
60 (orange bars). The purple bars show the number of cells
40
containing the same CDR3 clonotype of the respective receptor in the
# of cells

20
bulk sample.
10

0
IGH CDR3s

13
 Cell sorting and flow
cytometric quality control
Our solutions for gentle microchip-based multiparameter cell sorting can play
an important role in boosting genomic results and saving time and costs.
Flow cytometric quality control can further protect against wasteful analyses
and set expectations for results by providing accurate cell counts of viable cells.

14
 FLOW CYTOMETRY

Gentle multiparameter cell sorting

Flow cytometric cell sorting enables the isolation Gentle cell sorting improves single-cell
of target cells with highest purities based on the
combination of different markers and physical gene expression analysis of murine
parameters, allowing also the simultaneous depletion mammary gland epithelial cells
of dead cells and debris. However, the sorting process MACSQuant Tyto ensures the gentle cell sorting
using droplet-based sorters can damage the cells due of cells, such as mammary gland epithelial cells, to
to high pressure and electrical charges within the minimize transcriptional changes and obtain samples
system, and lead to the upregulation of stress-related of high quality to result in more reliable single-cell
genes. The MACSQuant® Tyto® Cell Sorter allows genomic analysis.
gentle multiparameter cell sorting using a unique
microchip-based technology, avoiding harsh sorting
conditions and thereby resulting in samples of A nGene nUMI
highest quality. 1,191 1,431 2,393 3,188
6,000
30,000
The actual sorting process takes place within the
MACSQuant Tyto Cartridge, a single-use and fully 4,000
20,000
closed system that eliminates the risk of sample
contamination and carry-over as well as the release 2,000 10,000
of aerosols. In addition, during the sorting process,
target cells are highly concentrated, which might
0 0
supersede additional centrifugation steps after the Droplet MACSQuant Droplet MACSQuant
cell sorting process. Sorter Tyto Sorter Tyto

B
4
3

0
0.1 7

0.1 6
0. 5

0.1 5
0. 3

57
0.1 2

54

11
84

66

56

38

4
0
2
9
3

08

89
65
87
45
33

33

12
08

09
32
24

MACSQuant
0.1

0.1

0.1

0.1
0.

0.

Tyto
0.1 4
4

0.1 1
0. 4
0. 4

0.1 2
0. 6

20
0.1 5

0.1 0
12
08

80
30
31

80

8
9
9

99
58

07

38

25
42

15

Droplet
08

20
26

33

21
0.1

0.1
0.

0.

Sorter
o l re
o n o es
D N x i d r ia l osis

m st s
e ss

Au cro r
ph s
fo H m l d y
e d o ti c th

ei n s
sp g
se
N e p ai
to s i

o t si g e s
d a ve e

Os Cel ag

re in
a g re
m at u

ld y p o ea

on
ch A p cu l

A at i e n

p r x ia s tr
n al
pt

re
g
e
i o ign
es g s

O d
n
d h lin
ll a n d

i to
Ce l ha

M
l
Ce

Un

Percentage of cells
expressing the gene 40 50 60 70 80

Figure 15: Gentle cell sorting improves single-cell gene expression

analysis of mammary gland epithelial cells. Epithelial cells from
mouse mammary glands were sorted using a droplet-based sorter
or the MACSQuant Tyto. Sorted cells were then applied to the
10x Genomics Chromium™ Platform. (A) An increased number of genes
and unique molecular identifiers (UMI) was obtained from cells sorted
with a MACSQuant Tyto compared to a conventional droplet-based
sorter. (B) Cells sorted using MACSQuant Tyto showed lower expression
of stress-related genes compared to a conventional droplet-based cell
sorter. Dots colored in red indicate which data set shows higher gene
activation. The dot size indicates the percentage of cells expressing
the respective gene. Data kindly provided by Quy Nguyen, University
of California, Irvine.

15
FLOW CYTOMETRY

Decrease the time needed for flow

cytometric cell sorting and analysis
The frequency of specific cell populations in certain
samples, such as tumors, can be very low and lead
to extended duration of sorting and acquisition
times. Pre-enrichment of these cell populations
by immunomagnetic cell isolation with MACS®
MicroBeads can significantly save time and increase
the quality of your flow analysis or flow sorting.

Cell type Cells to Events to Flow Total flow

analyze collect cytometry cytometry
time/sample* time**

CD4 + T cells
Bulk 5,000 7.96×10⁶ 66.3 min >10 h
Isolated*** 5,000 5.41×10⁴ 0.5 min ~11 min

CD8+ T cells
Bulk 5,000 2.80×10⁶ 23.3 min >3.5 h
Isolated*** 5,000 4.37×10⁴ 0.4 min ~10 min

T cells
Bulk 10,000 8.13×10⁵ 6.8 min >1 h
Isolated*** 10,000 3.24×10⁴ 0.3 min <10 min
* Flow rate: 2,000 events/s
** C onsidering 9 samples (3 experimental groups × 3 replicas/group). Includes 45 s
automated mixing and rinsing between samples on the MACSQuant® Analyzer
*** I solation using CD8 (TIL), CD4 (TIL), or CD4/CD8 (TIL) MicroBeads, respectively

Table 3: Cell enrichment helps to decrease time for flow sorting

and analysis. Isolation of CD4 +, CD8+, and pan T cells from different
mouse tumor models using mouse CD4 (TIL) MicroBeads, CD8 (TIL)
MicroBeads, and CD4/CD8 (TIL) MicroBeads dramatically decreases
time of analysis.

16

Quality control
Quality control of samples prior to downstream
genomic analysis can save time, costs, and set the
right expectation for the outcome of the analysis.
Especially when performing single-cell genomic
analyses, there are certain requirements a sample
must meet to achieve reliable, reproducible, and
accurate results. These requirements include having
cell viability above 90%, being in a single-cell
suspension, and having an accurate cell count.
The MACSQuant® Analyzer Flow Cytometers
allow you to check all these parameters at once.
The MACSQuant Analyzers make flow cytometry easy
for any user. With their unique function of volumetric
pipetting, absolute cell counting is automatically
provided with every sample measurement and every
Total events acquired
55%
Total cells
Side scatter-A
PI
Forward scatter-A
Figure 16: Easy and fast assessment of cell count and cell viability before downstream genomic applications. Exemplary quality control analysis
of a neural cell suspension obtained after dissociation of mouse brain and analyzed using a MACSQuant Analyzer 10. Cell viability was determined using
the DNA intercalating dye propidium iodide (PI) and the autolabeling function of the instrument.
FLOW CYTOMETRY
population being analyzed without the need for
counting beads and in less than a minute. As little as
25 µL of your sample is enough to obtain an accurate
total cell count and determine the viability of your
cells of interest. Furthermore, the Count and Live Cell
Discrimination Express Modes enable you to obtain
the results of the number of viable cells present
in the sample automatically.
There are many other innovative and beneficial
features of the MACSQuant Analyzers. For example,
the autolabeling function for full automation of
sample acquisition and the statistic reports available
with the integrated software package.
Gated on total cells Gated on viable cells
Forward scatter-H
99%
96%
Viable single cells
Viable cells
3.03×10⁴ cells/mL
17
 Tips and information
Our recommendations for the preparation of cells
and nuclei for genomic analyses.

18
Best practice tips Product information
• Prepare your samples as fast as possible. Product Order no.
Minimize cell preparation steps to avoid cell death Instruments
and changes in the gene expression pattern. gentleMACS Octo Dissociator with Heaters 130-096-427
• When possible, use wide-bore pipettes autoMACS Pro Separator Starter Kit 130-092-545
to minimize cell damage and use nuclease-free MACSQuant Tyto Cell Sorter 130-103-931
reagents and consumables. For more information about the MACSQuant Analyzers Flow
• Some cells tend to aggregate after tissue Cytometers visit miltenyibiotec.com/macsquant4genomics
dissociation due to free floating DNA. Reagents
An additional DNAse wash after dissociation MACS Tissue Storage Solution 130-100-008
might help reduce cell aggregates. Dead Cell Removal Kit 130-090-101
• Before starting your single-cell analysis, obtain Debris Removal Solution 130-109-398
the total cell count of your final suspension and Red Blood Cell Lysis Solution (10x) 130-094-183
analyze cell viability. Nuclei Extraction Buffer 130-128-024
• Remove dead cells when viability is below 90% For a complete list of Tissue Dissociation Kits, visit
miltenyibiotec.com/TDK4genomics
to increase the recovery of viable cells.
For a complete list of MicroBeads and Isolation kits, visit
• Increase the recovery of nucleated cells by removing miltenyibiotec.com/kits4genomics
red blood cells. However, red blood cell lysis may For a complete list of StraightFrom products visit
induce cell stress responses. Therefore, apply miltenyibiotec.com/SF4genomics
red blood cell lysis only if necessary. For example, For more information about products for direct isolation
if target cells are isolated, removal of red blood cells of PBMCs from blood contact us

may be dispensable.
• Remove debris and cell clumps from your sample VISIT
before analysis. This allows for more accurate
Find more downloadable application
cell counts and avoids clogging during cell sorting,
data here
as well as on the single-cell genomics platform.
 iltenyibiotec.com/
m
• Apply cell or nuclei suspensions to your single-cell data2download
genomics platform immediately after preparation.
Storage may lead to clumping of cells or nuclei.
If storage is unavoidable, store samples on ice for
a short period of time, not exceeding 30 minutes.

19
 miltenyibiotec.com/cellprep_genomics

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