Methods for CD8 depletion in mice.

[Craig Walsh]

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.

Craig Walsh