T15: Cell Separation
When cells do one have a high enough PE, physical or immunological separation may be
needed. Separation techniques give a higher yield quicker, but are not as pure.
Separation techniques depend on variations in:
Antibody affinity to cell surface epitopes
Light scatter/fluorescent emission when sorted by flow cytometry
15.1 Cell Density and Isopyknic Sedimentation
Isopyknic means “of the same density”
Centrifuging is used to separate cells of different
density. The cells will separate
Protocol 15.1 outlines how to separate cells by centrifuging: form gradient, centrifuge,
collect fractions, dilute, culture.
Variations of Protocol 15.1 include position of cells, other media, and
use of marker
beads. Marker beads can be used to establish different density areas of the gradient.
Isopyknic sedimentation is faster and gives higher yields than velocity sedimentation. It should
be used when there are clear differences in density.
Cell Size and Sedimentation Velocity
A relationship exists between particle size and sedimentation rate, and is given
15.2.1 Unit Gravity Sedimentation
If you layer cells over a serum gradient, the cells will settle by
the equation given above.
There is a caveat, unit gravity sedimentation is limited to smaller numbers of cells.
15.2.2 Centrifugal Elutriation
A centrifugal elutriator increases sedimentation rate by separating cells in a centrifuge
specifically made f
or separation. Basically, cells are pumped into a separation chamber, which
pushed them to the outer edge. Medium is pumped through to balance the sedimentation rate of
the cells. Because of the differences among cells, sedimentation occurs at different ra
tapered shape of the chamber means there is a gradient of flow rates.
Wow, this seems like a very cool machine. It does seem prohibitively expensive though for a class
to use just a couple times. The cost could probably be justified in a lab that needs to separate
cells a lot.
d techniques depend on antibody specific binding to an epitope.
15.3.1 Immune Panning
Immune panning: attaching cells to dishes coated with antibodies. The cells
targeted by the antibodies will attach at the bottom of the cell.
This technique uses antibodies against a cell surface epitope that are conjugated to micro
or ferritin beads. The cells are mixed with the beads and placed in a magnetic field. The cells
attached to the beads will separate.
Protocol 15.2 outline
s magnetic sorting. Mix cell suspension with antibody
and place in magnetic separation column. Bound cells will stay in the column and unbound cells
will flow through. Bound cells are purged with a syringe piston.
vated cell sorting
This technique uses a laser beam so that cells will scatter light. A flow cytometer
measures photomultipliers. A fluorescence
activated cell sorter will use the emission from each
cell to sort it into sample tubes or waste container.
remember lasers being used in plant tissue culture techniques. It’s cool that they have so many
15.5 Other Techniques
Some other separation techniques are: electrophoresis, affinity chromatography, and.
t know electropohoresis could be used to separate cells. I wonder how large the pore size
would have to be for the gel.
15.6 Beginner’s Approach to Cell Separation
It is recommended to start simply, with techniques like density gradient centrifugation. I
high purity is needed, a minimum of a two
step fractionation is required.
16.1 The Need for Characterization
There are 6 requirements for characterizing cell lines:
Show there is no cross
Confirming species of origin
Correlating with tissue of origin
See if cell line is transformed
Does the cell line have a tendency for phenotypic variation or genetic instability?
Identify specific cell lines
16.2 Record Keeping and Provenance
Record keeping/provenance is important, especially if a cell line proves to be valuable.
Many cells commonly used are cross
contaminated, which is one reason why
characterization is so important.
I wonder who big of a problem cross
contamination is. The text seems to imply that it is an
unrecognized but major one.
16.3.1 Species Identification
Karyotyping: chromosomal analysis
Karyotyping is important in confirming the species of cells.
16.3.2 Lineage or Tissue Markers
l which tissues cell lines come from, cell surface antigens, intermediate filament
differentiated products/functions, and enzymes are used.
16.3.3 Unique Markers
Other markers include chromosomal abnormalities, MHC group antigens, and DNA
Transformation is such a large topic that it is discussed in Ch 17.
16.4 Cell Morphology
Monitoring morphology is the easiest way to identify cells, but has some deficiencies.
One of which is how cell morphology chan
ges depending on culture conditions. Despite these
shortcomings, frequent observations of cultures are more useful than occasional stains. Recall
from chapter 13 that unhealthy cells will become granular and show vacuolation around the
The inverted microscope is often used incorrect
contrast increases contrast of
Protocol 16.1 outlines how to use an inverted microscope. Place the culture on the microscope
stage, choose the correct optics. Focus and c
I would have assumed that using a microscope is a basic task, but I suppose it doesn’t hurt to
cover the basics.
Giemsa is one way to easily prepare a stained culture.
Protocol 16.2 shows how to stain with Giemsa: fix culture in
methanol, stain with Geimsa, and
dilute to 1:10. Wash and examine.
16.4.3 Culture Vessels for Cytology: Monolayer Cultures
Petri dishes, coverslips, microscope slides, OptiCell, and petriperm dishes are all suitable
16.4.4 Preparation of
Suspension Culture for Cytology
Cells from a suspension culture must be places on glass/plastic for cytology. Centrifuging
is the preferred way to create monolayers from suspension.
Protocol 16.4 shows how cells can be centrifuges on a slide.
uging sounds so cool!
To record images from a microscope, use a CCD camera. Film cameras are not
recommended, as digital cameras are cheap and produce images that are easy to store.
16.5 Chromosome Content
is one of the easiest ways to identify cell lines. Chromosome content can also
be used to differentiate between normal and transformed cells.
Protocol 16.7 outlines how to prepare for chromosome analysis. Fix cells in metaphase and
swollen in hypotonic me
dium. Stain and examine on slides.
16.5.1 Chromosome Banding
There are several techniques of identifying chromosome pairs with little morphological
differences. They include:
16.5.2 Chromosome Analysis
the following is how the chromosomes are analyzed:
It’s interesting how they use Photoshop to digitally compare chromosomes.
16.5.3 Chromosome Painting
By using fluorescent probes, genes, translocations, and species of origin
can be located.
16.6 DNA Content
Ways to measure to DNA: propidium iodide fluorescence and flow cytometry.
16.6.1 DNA Hybridization
Hybridization gives information regarding regions specific to species, amplifies areas of
DNA, or changed base sequen
16.6.2 DNA Fingerprinting
Minisatellite and microsatellite DNA are regions that are not transcribed. They can be
used in forensics due to differences in their length.
Protocol 16.8 outlines the procedure. Digest cells with restriction enzyme, perf
blot, and hybridize.
Fingerprinting is also useful in TC in that it can exclude cross
contamination and confirm
16.6.3 DNA Profiling
DNA profiling is only used in human now and is based on the fact that short tandem
repeats of mi
crosatellite sequences have been quantified.
Protocol 16.9 outlines how to perform DNA profiling.
16.7 RNA and Protein Expression
A Northern blot is a way to analyze gene expression. When extracts are run are 2
they produce a wealth of
information difficult to interpret. Using a computer to scan the gels
helps to produce the data efficiently.
16.8 Enzyme Activity
Some, but not many, enzymes will be expressed
. If using marker enzymes,
compare the uninduced and induced levels wi
Enzyme activity can also be studied by comparing cell strains among species.
16.8.2 Isoenzyme Electrophoresis with Authentikit
Authentikit screens 6 cell lines for 7 genetic makers.
ation can be determined through immunostaining. This method uses an
antibody conjugated to a fluorochrome, or depositing a product conjugated to an antibody. It can
be direct, which is when the antibody is conjugated to the fluorochrome/enzyme and stains
irectly. Or it can be direct,
in which the primary antibody binds to the antigen. Then there is a
second antibody against the Ig of the first antibody. The second antibody is conjugated with
I wonder why indirect is more common. It sound lik
e it would be more complicated to have two
antibodies instead of one.
Assaying quantifies marker and protein expression. They can be done through kits.
Many characteristics describes above are signs of diff
erentiation. Chapter 3 gives other
examples of differentiation
What 4 differences among cells do separation techniques depend on?
Cell density, antibody affinity, cell size, and light scatter/fluorescent emission
What is the
based techniques depend on antibody specific binding to an epitope.
What is immune panning?
attaching cells to dishes coated with antibodies
Pro/con of using cell morphology as a way to identify cells?
Pro: easy; con: ce
ll morphology is plastic
What do assays provide?
A quantifiable measure of marker and product protein expression