Wed Jun 01, 2022 2:51 pm by mleipold
Hi all,
I would point out that there are a few reasons to have controls in your experiments.
1. Make sure all your *probes/antibodies* are working properly (including whether you accidentally left out one of the 30-40 antibodies when making the cocktail.....). In this case, a single control (Unstim, or a single Stim) might be sufficient per batch, depending on your experiment. And, if your probes aren't sensitive to fix, you may even be able to pre-make Unstim and Stim cell aliquots so that you'd have enough for an entire study. I think we've had a study where the researchers got Biolegend to make Fixed Unstim and Fixed Stim Vericells that were enough for an entire study.....you might reach out to your rep.
2. Make sure that all your *reagents* are working properly. I've seen cases where a stock of PMA or LPS was working fine one week, and then something happened that caused it to stop working properly. In this case, if you were relying on pre-made Stim samples, you might not catch that your Stim reagents stopped working properly.
Therefore, in an ideal setting, you would have a control donor (PBMCs, or whatever your study samples are) that you treat exactly the same as all your study samples for *each staining set*: thawing, washing, counting, any rests or stims, etc. If you're running individual samples and not freezing them at some point, you may wind up running proportionately more controls than you would if you were doing larger barcoded sets (particularly if you were doing FBS/DMSO freezing at the end so you could stain multiple Plates in one experimental Batch).
However, there may still be ways to cut down on the total number of controls: for example, we've run some PhosphoPBMC sets where we thawed enough samples in one to fill two plates, but only did one control set. In this case, we did everything in parallel for the two plates up through and including the MeOH at -80C, and finished processing each plate separately. Formally, this only controlled for the stims (since both plates were stimmed together), not for the antibodies (at least the intracellular were stained in separate plates), but was considered to be a happy medium.
Mike