ATC : Lecture 4

Cell Separation
Cell Quantification
Cell Characterization
 Cell Separation
- Cloning and using selective culture conditions
are the preferred methods for purifying a culture
- However,
Cells with low platting efficiency
appropriate selection conditions are not
available

Need a physical or immunological separation

techniques
give higher yield more quickly
- successful separation techniques depend on
differences in :
a) Cell density (specific gravity)
b) Affinity of antibodies to cell surface epitopes
involve a relavely low level of technology and are
inexpensive
c) Cell size
d) Light scatter or fluorescent emission sorted
by flow cytometry.
involve a high level of technology with signicant
outlay of capital
 Separation Techniques

1) Centrifugation
a) Isopyknic sedimentation ( cell density)
b) Velocity sedimentation ( cell size)

2) Antibody-based techniques
a) Immune panning
b) Magnetic sorting

3) Fluorescence-activated cell sorting

 Antibody-based techniques

- Rely on the specific binding of an antibody to the

cell surface

Immune Panning
- Attachment of cells to dishes coated with antibodies

- A cell-type-specific antibody raised against a cell

surface epitope is conjugated to the bottom of a petri
dish
 Magnetic Sorting
- Uses specific antibody, raised against a cell surface
epitope, conjugated to ferritin beads or micro beads

- The cells that have attached to beads are drawn

to the side of the separating chamber

- The cells and beads are released current is

switched off

- Cells are separated from the beads trypsinization

or vigorous pipetting
Separating
chamber
(magnetic field)

http://www.miltenyibiotec.com/en/NN_736_MACS_Cell_Separation_the_principle_1.aspx
 Fluorescence-Activated Cell Sorting

- projecting a single stream of cells through a laser

beam in such a way that the light scattered from the
cells is detected by one or more photomultipliers
and recorded

- If the cells are pretreated (propidium iodide/

fluorescent antibody) emission excited by the laser
is detected by a second photomultiplier tube
 Fluorescence-activated cell sorter (FACS)
- Instrument that uses the emission signals from
each cell to sort the cell into one of four sample
collection tubes and a waste reservoir
- Used to separate cells according to any
differences that may detected by light scatter
(e.g. cell) or fluorescence (e.g. DNA, RNA, protein
content)
- Been applied to wide range of cell type utilized
most extensively for hematopoietic cells
http://galinirmal.wordpress.com/
http://www.antibodies online.com/resources
/17/607/Flow+cytometry+FACS+Principle+and+experimental+setup/
 Other Techniques
1) Electrophoresis
- based surface charge
- performed in Ficoll gradient or by curtain
electrophoresis
2) Affinity chromatography
- based on cell surface antigens or cell surface
carbohydrates
- e.g. antibody, plant lectin that are bound to
nylon fiber, Sephadex
- useful for fresh blood, less so for cultured cells
3) Countercurrent distribution
- based on affinity of cell surface
constituents for solvent phase
- To purify murine ascites tumor cells with
reasonable viability
* Murine : rodents e.g. mice/rat
 Cell Quantification
Purpose
1. Characterization of the growth properties
of different cell lines

2. Experimental analyses

3. To establish reproducible culture conditions

for the consistency of primary culture

4. maintenance of cell lines

 Cell Counting
Hemocytometer

- Placing the cells in an optically flat chamber

under a microscope
- The cell number within a defined area of known
depth is counted and the concentration is derived
from the count
- Advantages cheap, can see cells and viability
stain can be used
- Disadvantages high stascal error, slow and
low sensitivity
Electronic counting
Three main types:
1) Resistance-based cell counter
- Based on the change in current generated
when a cell passes through a narrow orifice
- e.g. Beckman Coulter Z1, Z2 or Innovatis CASY
2) Image analysis cell counter
- A microscope view of unstained or stained
cells/nuclei in special counting chambers by
visible light/ fluorescence
- e.g. Invitrogen Countess
3) bench-top flow cytometers
- Analyze a single cell stream for cell
concentration and other parameters
- e.g. Guava, Accuri

Advantages quick, large sample counted, low

error and sizing possible
Disadvantages blind; counts dead cell and live
cells and clumps, coincident
signals at high cell concentrations
CASY Electronic cell counter

C6 Accuri Cytometer

Countess (Invitrogen)
 Cell Characterization
Main Requirements
1) Authentication
confirmation that the cell line is not cross-
contaminated or misidentified
2) Confirmation of the species origin
3) Correlation with the tissue of origin
Identification of the lineage to which the cell belongs
Position of the cells within the lineage
4) Determination of whether the cell line is
transformed or not
Finite or continuous cell line?
Does it express properties associated with
malignancy?
5) Indication of whether the cell line is prone to
genetic instability and phenotypic variation.
6) Identification of specific cell lines within a group
from the same origin, selected cell strains, or hybrid
cell lines
 1) Cell Morphology
- simplest and direct technique used for cell
identification
- Disadvantages :
plascity of cellular morphology in response
to different culture conditions.
alteraon in substrate and the constuon of
the medium can also effect cellular morphology
- comparative observations of cells should always
be made at the same stage of growth and cell
density in the same medium, and growing on the
same substrate
 Examples of cell morphology in culture

OP9 cell = mouse stromal cell

Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny
Maureen R. Lynch1, Judith C. Gasson2, Helicia Paz1
1Department of Medicine, Hematology-Oncology, University of California, Los Angeles, 2Department of Biological Chemistry, University of California,

Los Angeles
 Microscopy
Inverted microscope most important tools in
observation of cell lines

- photographic records a set of photographs at

different cell densities for each cell line
used as a record in case a morphological change
happen
 Staining
Giemsa convenient method of preparing a
stained culture.

gives a good high-contrast polychromatic stain

Pink/magenta = nucleus;
Dark blue = nucleoli;
Pale gray blue = cytoplasm
Morphology of Bone Marrow by Giemsa staining

Pink/magenta = nucleus
Dark blue = nucleoli
Pale gray blue = cytoplasm

Haddad, A.S. et al. Haematologica 2008;93:e1-e5

 Photomicrography

-recording microscope imaging digitally

CCD camera.
- give advantage of constant monitoring and
connection to an intranet for file storage, printing
and accessibility
 2) Chromosome
- Identifying cell lines and relating to the
species and sex from which they were derived.
- can distinguish between normal and malignant
cells.
Chromosome Banding
- enable individual chromosome pairs to be
identified when there is little morphological
difference between them.
- Banding pattern is characteristic for each
chromosome pair
 Methods
Giemsa banding (G-banding)
- chromosomal proteins are partially digested by
crude trypsin, producing a banded appearance
on subsequent staining
Q-banding
- stains cells in 5% (w/v) quinacrine dihydrochloride
in 45% acetic acid
C-banding
- emphasizes the centromeric regions
 fluorescent staining with Hoechst 33258
and alkaline staining with Giemsa
- For discriminating between human and mouse
chromosomes to aid the karyotypic analysis of
human-mouse hybrids

mouse centromeres will uoresce more

brightly than human centromeres
 Chromosome banding

Normal Human chromosomes

(G-banding staining) http://www.sciencephoto.com/media/312936/enlarge
 Chromosome Painting

- To overcome traditional banding techniques which

cannot characterize many complex chromosomal
aberrations
- new techniques spectral karyotyping (SKY) and
multicolor fluorescence in situ hybridization (M-
FISH)
- allow the simultaneous visualization of all 23
human chromosomes in different colors
 Chromosome Painting (M-FISH)

Premature aging disease

(Werner Syndrome)

Alteration of
chromosome 6 and 10

http://www.austincc.edu/mlt/mdfund/mdfund_unit10pictures.htm http://www.salk.edu/news/pressrelease_details.php?press_id=286
 3) DNA Analysis
DNA content
- measured by propidium iodide fluorescence with a
CCD camera OR flow cytometry.
- DNA can be estimated in homogenes with DNA
fluorochromes e.g. DAPI, Hoechst 33258, Pico Green
- amount of DNA per cell is relatively stable and is
the characteristic to the species in normal cell lines.
- useful in characterization of transformed cells
that are often aneuploid and heteroploid.
 DNA Hybridization

- hybridize specific molecular probes to unique

DNA sequences
- detect the hybrids by radioisotope, fluorescent
or luminescent labels.
- provide information about species-specific
regions, amplified regions of the DNA, or altered
base sequences that are characteristic to that cell
line
 DNA Fingerprinting
- powerful tool to determine the origin of a cell line
DNA from the cell or from other donor
individual has been retained.
- appear to be quite stable in culture, and cell lines
from the same origin
- Methods :
Restriction fragment length polymorphisms (RFLPs)
- very accurate but it requires a considerable
amount of DNA to be used
Restriction fragment length polymorphisms (RFLPs)

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 DNA Fingerprinting

Variable Number of Tandem Repeats (VNTR) pattern analysis

 Short tandem repeat (STR)

- the most popular and innovative DNA

fingerprinting method (forensic cases and
paternity testing)
- works by analyzing the DNA variations between
individuals (polymorphism)
Other related techniques :

4) RNA & Protein Expression

- RNA expression can be quantified by reverse transcriptase PCR
(RT-PCR)

- Protein can be detected by microarray analysis using antibodies

5) Enzyme activity
- specific marker enzyme

6) Antigenic markers
- Immunostaining and ELISA assays