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.

savagecowcreekMécanique

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

82 vue(s)

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


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