Re: low antigen-specific CD4 responses from frozen PBMC

Has anyone had any experience with N418-biotin (hamster-antiCD11c) on porcine PBLs? 

thanks

jody

>>> "Joshua Walker" <walkejos@ohsu.edu> 12/06/04 05:37pm >>>
  I have seen this problem before as well.  In our lab we frequently 
work with frozen PBMC and get reproducible and robust CD4 responses.  
Our protocol is as follows...
1) Thaw cells in warm (37C) media
2) Wash cells twice with warm media
3) Stimulate for 6hrs immediately (I think this is where you are 
running into trouble.  I have seen greatly diminished CD4 responses 
when we have allowed the cells to "rest" overnight.)
4) Hold samples at 4C overnight
5) Stain
Hope this helps.

Josh Walker


On Dec 5, 2004, at 11:31 AM, Elizabeth Sinclair wrote:

> Hi
> We have a CFC/ICS problem with frozen PBMC. We recently compared the 
> CD4 and CD8 responses of frozen PBMCs stimulated with pp65 and p55 
> peptide pools to whole blood responses previously obtained in the same 
> subjects. While the CD8 responses correlated well, the frozen CD4 
> responses were reduced or lost completely. CD4 SEB responses were 
> maintained in frozen PBMCs, usually higher in frozen than fresh whole 
> blood.
>
> We are using the BD protocol for frozen PBMC CFC:
> Thaw, plate, rest overnight, stimulate 6 hours, hold at 18oC, EDTA, 
> FACs lyse, FACs perm, stain etc.
> We also do a EMA step before FACs lyse.
> We have tried some modifications based on other published methods:
> 1. Thawing in to cold media vs thawing in to a large volume of warm 
> media - no difference
> 2. Stimulating at 37oC overnight vs 37oC for 6 hrs followed by 18oC 
> hold - first experiment overnight stimulation looked better for CD4 
> cells but this was not repeated in subsequent experiments.
> 3. FACs lyse and FACs perm vs FACS lyse with tween 20 for fix and perm 
> - No consistent difference.
> Cells that are frozen by our AIDS specimen bank and in our lab give 
> the same results, so I don't think it is a freezing problem, unless we 
> are both doing something wrong.
> Any suggestions as to how we might be losing CD4 antigen-specific 
> responses would be much appreciated.
> thanks
> Elizabeth