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ELIspot and PBMC culture


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#1 ehcw

ehcw

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Posted 14 July 2010 - 08:09 PM

Hi there,

I am doing some IFNg ELIspot with human PBMCs to test out their reaction to some of the peptides I designed and I have encountered a few problems that I would like to have some advice on.

1) The PBMCs were isolated using the ficoll-plaque method, stored in liquid nitrogen suspended in R20 medium (RPMI-1640 + 20%FBS + L-Glu + Pen/Strep + 10%DMSO). There was an accident with LN and now a lot of PBMC died or look very unhealthy (weird shapes). I am hoping a little culturing of PBMC will increase the number or at least make them healthier. I can't seem to find a standardized protocol for culturing PBMCs. I am thinking about using R10 (same as above but without DMSO and only 10%FBS) to culture them, is that a good idea? I am a little bit hesitated about adding IL-2 as I don't know if they will activate the PBMC and affect my result with other peptides on ELIspot. Anyone has a good detailed protocol or advices to share?


2) The peptides, after reconstituted, contain a bit of DMSO. I am curious that if the DMSO will affect the viability of the PBMCs. Do you do any "wash" to the peptides before put it together with the PBMCs on the ELIspot plate?

3) If you are getting very little spots, do you usually do a pre-treatment on a different plate (indirect method) or do a longer incubation on the ELIspot plate? Does it matter?

Thanks in advance,




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