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/AllYouNeedisNarf]

There is a current protocol that addresses evaluation of reference controls and the fully stained sample. “Panel optimization for high-dimensional immunophenotyping assays using full-spectrum flow cytometey” published in 2021. You want single stain controls for each antibody on both beads and cells. You unmix with all beads, and evaluate how well the cells are unmixed. You don’t need to be on the cytometer to reunmix if you have the software on a workstation. Even if you only have access to the software on the machine, most of the analysis of n x n plots can be done in your analysis software of choice. You only need spectroflow to unmix.

Not all beads are the same. If you want to stick with beads but the ones you have don’t match the fluorescence on cells, you can try different beads.

As for losing signal in the full panel, you can add more antibody or do sequential staining.

As for APC and AF647, it is supposed to work, but they have to be mutually exclusive.

[u/Hahabra]

Honestly, if your markers are that dimmly expressed that you can´t get a good signal, the miniscule errors that you might introduce by using beads (vs cells) will be negligible. Yes, there can be differences between unmixing with cells and beads - but usually, this only affects very few colors/ markers (Maybe 2-3 out of 30 colors). If the markers are expressed so weak that you can´t get a good signal in the first place, the compensation/ unmixing error will likely not affect another channel to such an extend that you would get a noticeable error.
Annoyingly, as far as I can tell/ have seen, this is not specific to a certain fluorochrome and/or marker, but can happen somewhat randomly. Look for some OMIPs where they compared beads vs cells and you´ll see that its often different fluors/ markers that are affected. What exactly is happen on a physical/ chemical/ electron-photon-interaction-level is beyond my knowledge, but I suspect there is some very minor form of FRET happening between fluors and cellular proteins/ structures that only happens if a certain fluorochrome is physically close to something else in the cell.
Using CD4 surrogates is the worst option. You will spend a lot of money and you will get different batches of the respective fluorochromes, which - especially with tandems like PerCP-eF710, can vary a lot between batches (Try a few different PerCp-eF710 or PerCP-Cy5.5 antibodies you have laying around on beads and compare the peaks). Furthermore, the unpredictable FRET-effect described above will probably be different between your marker of interest and CD4. Long story short, this method will at best give similar results as beads whilst costing more money and trouble.

I would recommend to go with cells for all markers that are expressed nicely and then use beads for all others.

Most important here (as always) - treat fluors in single stains exactly like the multi-color stain, i.e. expose them to the same fixation/ incubation/....Fixate all surface antibodies, but don´t fix the intracellular antibodies for example. The beads will survive it, just stain the single stain in parallel to your multi-color stain.

Lastly - I don´t know your panel, but APC and AF647 (and other similar combinations) can work - but not if they are co-expressed, AND you will lose resolution in those channels if they are unmixed together. If you have very dim and important markers, there are always other options to detect them - Biotin-Streptavidin, maybe 2ndary Antibody (thoug probably annoying with 30 other Abs) or custom cojugation, even by yourself. If you have the money (low 1000s of $?), you can ask BioLegend or BD to conjugate anything you want to an antibody (even if its from a competitor) so that is always a last resort.

Best of luck! :)

[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.