Cytokine Assay: supernatants to pool or not to pool? - (Jan/06/2012 )

Hi all;

I am new (both Forum and cytokines!!).

We are trying to detect cytokines from cultured/stimulated PBMC.
We are incubating in triplicate for 5 days.

Do you pool your supernatants or Not?

Currently, we are not pooling the supernatant and running each individual well with a CBA and we are observing quite variations between wells.
We only run them once.

So what are your best practices?

Pooled supernatants and run in duplicates?
Unpooled supernatants and run once?


Thank you very much for your help!!!!

Hi

I wouldn't recommend you to pool your samples as this could mess your results while analyzing. I culture BMDM in triplicate usually in a 24-well plate, and then I use the supernatant of each well to proceed with cytokine assay (ELISA). If you didn't do anything wrong the results should be somewhat similar but they will differ, hopefully your SD won't be too high.

If you're afraid of too many freeze-thaw cycles just make aliquotes of your samples to be loaded in future assays.

Hope this helps.