Methods for CD8 depletion in mice.

[JAF at UNCVX1.OIT.UNC.EDU]

Did you test the antibody for activity after you purified it.  Lots of 
antibodies die when ppt by NH3SO3.


On Thu, 6 Apr 1995, Craig Walsh wrote:

> 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
> 
>