Methods for CD8 depletion in mice.
walsh at biovx1.biology.ucla.edu
Wed Apr 5 20:58:35 EST 1995
Hello all. I have been trying to use the various published
protocols to deplete CD8 cells in mice. Basically, I have grown
up a big batch of Lyt2.43 that we got from ATCC, did an ammonium
sulfate precipitation (a 25:1 cut) followed by dialysis (1000:1).
I have injected 0.3 ml (appr. 2 mg) of "protein" into mice at
day 0, day 2 and day 4, and tested lymph nodes for CD4 and CD8
ratios, comparing them with mice that were injected with an
irrelevant antibody, and those without an antibody injection. I have
not seen any depletion whatsoever. On the other hand, I did the
exact same thing with the anti CD4 antibody GK1.5 and got almost
complete depletion of the CD4 population.
What am I doing wrong? Is there some little trick to this? The
hybridomas were grown in 500 ml of DMEM with 2% FCS/CS and grown
to a density of about 4 x 10^5 cells/ml (this was the maximum
density, at which point the cells started to croak). Anyone got
any good antibody that they would be willing to trade/sell? I'm
getting frustrated! :(
Thanks for your suggestions and help.
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