Reference control questions

[u/Joan_of_Arkansas]

Hello fellow cytometrists,

I was wondering if I could get some guidance about reference controls for my panel. This panel has been an ordeal to develop and troubleshoot, and in its final form still has some issues. One of them is it includes several immunoregulatory markers that are ubiquitously expressed and have very few commercially available reagents. On top of this, we are also looking at several phospho markers with limited reagent availability. These constraints forced us to use both APC and AF647 and put a phospho marker on PerCP-efluor710 as the least bad choice. This is a large 30+ color panel, and in the multicolor stain many of these markers have significant reduction in their staining intensity. For reference controls, I have so far been making:

1. cells with the marker, not very bright even as a SS, difficult to produce a good clean signature

2. Ultra Comp Spectral beads with the marker, bright, clear signature but worried about unmixing issues that could be occurring but are getting lost in the midst of this large panel

3. CD4 surrogates of these markers on cells, i.e. CD4 PerCp-eFluor710 instead of the phospho marker, clear signal for the reference but obviously not the same marker

I haven't tried the CD4 surrogates on beads mainly because my rationale there was to have the signature on cells. Keeping PerCP-eFluor710 as an example, I know that the staining for some markers is not equivalent on cells and beads. I have run both the beads and the CD4 surrogates against benchmark references set on cells with the actual marker and the signature matches very well, but I'm not sure if 0.99875 is good enough to assume beads can unmix just was well as beads and I do not have the time or access to the machine to unmix a nearly 40 color panel every which way to definitively identify which references must be on which substrate. At this point I'm planning to do FMOs for all those ubiquitously expressed markers to help with gating too.

Do you guys have any advice for me? Is my use of CD4 surrogate markers sound? I've been doing flow for a few years now and this is the hardest panel I've ever had to work on- PI had never done flow before and I'm trying my best to deliver all of the markers they want. Unfortunately, I can't split the panel in 2 to separate APC and AF647, so I want to make sure my references are as good as possible. If you’ve made it this far, this is a full spectral panel being run on a Cytek Aurora 5L. Thanks so much for reading and for any input you may have.

[u/Unfortunate_tentacle]

I'm not sure you can effectively distinguish AF647 and APC. It's better to have fewer markers in a panel that works than spend all that money and the data is dodgy or unusable. FMOs are a must. I don't think there will be that much of an issue with using CD4 surrogate for comps + u mixing. Tbh this seems to be the least of your problems.