What's the best way to sort T cells?
ghermans at luc.ac.be
Fri Mar 28 10:04:22 EST 1997
In article <33379EB2.486A at west.bidmc.harvard.edu>,
mkoziel at WEST.BIDMC.HARVARD.EDU (margaret koziel MD) wrote:
> I would like to perform a pilot experiment in which I sort T cells into
> CD4,CD8, CD28, and possibly Cd45RO/RA. I know that FACS sorting will
> give me the best purity, but it would be fairly expensive for me to do
> this. What's your opinion regarding Ab coated magentic beads vs. the Ab
> coated tissue culture flasks that are available? I'm aiming for 90-95%
> Margaret Koziel
FACS is not only expensive, it's also tediously slow if you want to gather
a large number of cells. It doesn't work on populations below 1%, but
given your markers that won't be a problem.
Beads are easy and versatile (buy goat-anti-mouse coated ones, label
target population with whatever mouse monoclonal you need).
However, the cheapest way I ever came across was simply coating disposable
plastic petridishes with antibody (doens't even need to be very pure -
ascites will do). Also good to know: you can recover the
antibody-containing coating buffer and coat another petri dish using the
same dilution. After coating a plate, you incubate the cell suspention in
the dish at 4 degrees celcius (= the fridge) for some time, and gently
wash away the non-bound population. The tricky part is the washing:
impossible to control exactly, so the method is not very reproducible. But
then again, most of these techniques isn't...
Guy Hermans, PhD student
Ms research Unit Immunology research group
Dr. L. Willems-Institute Dept. of Physiology, LUC
University Campus University Campus
B-3590 Diepenbeek B-3590 Diepenbeek
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