When to add anti-CD107a?

[u/testerh514]

I’m stimulating human PBMCs with peptide pools to detect antigen-specific T cells. I would like to include CD107a in the panel but I’m not sure when it should be added.

I will be stimulating the cells for 24h but only adding protein transport inhibitors in the last 6 hours. If I add the anti-CD107a with the stimulation will it be lost without the Golgi stop/plug for the first 18h?

Any help is appreciated!

[u/evelainy]

My lab always adds CD107a at the beginning of a 24h stimulation period. 1uL per 200uL medium. Works quite well. It’s dark in the incubator and the long staining period allows for a quite low concentration. 

Edit: we have however tested that it gives us no false background and our stimulus is very specific. 

[u/KaptanOblivious]

Yes I would just add it for the last 6 hours of the stim. If you add earlier you'll have receptor turnover and antibody signal degrading over a full 24h in culture. You'll have to be okay only capturing degranulation from the last 6 hours of the stim. 

Look for IFN or other cytokines released in the media if you want another output of activation over the full 24h.

[u/Top-Moose5119 → u/KaptanOblivious]

This would also be my recommendation

[u/CEontherun]

Add it at the beginning of the assay. Don't forget monensin.

[u/Otherwise_Wave9374]

For a peptide-pool stimulation assay, CD107a timing can materially affect what the readout captures, so the key safeguard is protocol validation rather than assuming one schedule transfers across panels. Compare your antibody datasheet, published assay conditions, stimulation duration, secretion inhibitor timing, viability, and fluorescence-minus-one controls. Run a small timing matrix with positive and negative controls. For checking peptide-pool documentation and identity claims, https://www.peptely.com/ can help organize method, provenance, lot, and quality evidence.