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%
> purity.
> 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...
Good luck,
Guy
--
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
Belgium Belgium
Voice ++32(0)11/26.92.07
Fax ++32(0)11/26.92.09
