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.

lyricalwillingMécanique

22 févr. 2014 (il y a 3 années et 8 mois)

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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:

1)

Cell density

2)

Antibody affinity to cell surface epitopes

3)

Cell size

4)

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
in a
density gradient.

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.


15.2

Cell Size and Sedimentation Velocity


A relationship exists between particle size and sedimentation rate, and is given
approximately by:





v

r^2/4


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
tes. The
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.


15.3 Antibody
-
based Techniques


Antibody
-
base
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
that are
targeted by the antibodies will attach at the bottom of the cell.


15.3.2 Magneti
c Sorting


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
-
coated microbeads
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.


15.4 Fluorescence
-
acti
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.

I
remember lasers being used in plant tissue culture techniques. It’s cool that they have so many
applications.


15.5 Other Techniques


Some other separation techniques are: electrophoresis, affinity chromatography, and.
countercurrent distribution.

I didn’
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
f
high purity is needed, a minimum of a two
-
step fractionation is required.


F16 Characterization

16.1 The Need for Characterization


There are 6 requirements for characterizing cell lines:

1)

Show there is no cross
-
contamination

2)

Confirming species of origin

3)

Correlating with tissue of origin

4)

See if cell line is transformed

5)

Does the cell line have a tendency for phenotypic variation or genetic instability?

6)

Identify specific cell lines


16.2 Record Keeping and Provenance


Record keeping/provenance is important, especially if a cell line proves to be valuable.


16.3 Authentication


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


To tel
l which tissues cell lines come from, cell surface antigens, intermediate filament
proteins,
differentiated products/functions, and enzymes are used.


16.3.3 Unique Markers


Other markers include chromosomal abnormalities, MHC group antigens, and DNA
fing
erprinting.


16.3.4 Transformation


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
nucleus.


16.4.1
Microscopy


The inverted microscope is often used incorrect
ly. Phase
-
contrast increases contrast of
unstained cells.

Protocol 16.1 outlines how to use an inverted microscope. Place the culture on the microscope
stage, choose the correct optics. Focus and c
enter.

I would have assumed that using a microscope is a basic task, but I suppose it doesn’t hurt to
cover the basics.


16.4.2 Staining


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
for cytology.


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.

Cytocentrif
uging sounds so cool!


16.4.5 Photomicrography


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


Karyotype

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:

(1)

Giemsa banding

(2)

Q banding

(3)

C
-
banding


16.5.2 Chromosome Analysis


After banding,

the following is how the chromosomes are analyzed:

(1)

Chromsome count

(2)

Karyotype

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
ces.


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
orm Southern
blot, and hybridize.


Fingerprinting is also useful in TC in that it can exclude cross
-
contamination and confirm
identity.


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
-
D gels,
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
in vitro
. If using marker enzymes,
compare the uninduced and induced levels wi
th controls.


16.8.1 Isoenzymes


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.


16.9.1 Immunostaining


Antibody loc
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
d
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
fluorochrome.

I wonder why indirect is more common. It sound lik
e it would be more complicated to have two
antibodies instead of one.


16.9.2 Immunoanalysis


Assaying quantifies marker and protein expression. They can be done through kits.


16.10 Differentiation


Many characteristics describes above are signs of diff
erentiation. Chapter 3 gives other
examples of differentiation


Questions

1.

What 4 differences among cells do separation techniques depend on?

a.

Cell density, antibody affinity, cell size, and light scatter/fluorescent emission

2.

What is the
basis for
antibody
-
based techniques?

a.

Antibody
-
based techniques depend on antibody specific binding to an epitope.

3.

What is immune panning?

a.

attaching cells to dishes coated with antibodies

4.

Pro/con of using cell morphology as a way to identify cells?

a.

Pro: easy; con: ce
ll morphology is plastic

5.

What do assays provide?

a.

A quantifiable measure of marker and product protein expression