Personally, I have not found that including a viability stain for titrations was necessary. If you do have a high percentage of dead cells then you may find it beneficial to include a viability stain. I don't think people typically add in the viability stain with titrations unless they're struggling to interpret the data without it. The extra tools menu will let you unmix any number of fluorophores, just like the unmixing wizard in the acquisition tab.

@@UChicagoFlow Thank you very much for your expertise! Really highly appreciated! If I dare to ask, I would have 1 question concerning wet lab work. For the final staining, we would use 1 x 10^6 cells in 100 µl. For the titration, we would like to use less cells, eg. 100.000. Would you suggest to keep the volume constant (use 100.000 in 100 µl) or would you suggest to use less volume. Of course we would keep the final antibody concentration constant. I would really appreciate your help, since the literature on this topic is not quite clear!

@@Teresa-xt8oi There is a blogpost on the CAT Facility website with data supporting the idea of keeping the volume the same for your controls : voices.uchicago.edu/ucflow/2020/01/16/how-many-cells-should-i-stain-the-impact-of-cell-concentration-and-antibody-concentration/

@@chugcytometry3284 Thank you for your help!!

The overlooked point about the Spectral flow instruments is that you can, as you mention, just use the raw data without unmixing. You'd essentially be using the instrument as a traditional flow cytometer, and that is totally fine. The width of the bandpass of each detector is usually much smaller than what you'll find on you typical flow instrument, so fewer photons will hit the detector. But if the signal is bright enough, it should not matter.

@@chugcytometry3284 Thank you for the helpful response! I just titrated antibodies using mouse lung and was worried why a few populations hadn't resolved (CD206 BV605 and Siglec-F BV510). I'm hoping that it's a brightness issue that will be resolved once I go back and properly unmix the files this week.

This question comes up a lot, and I don't think theres a consensus on which way is best. I think a lot of people end up trying both ways.

@@kaylaparr7732 Do you mean that you don't have a positive signal at all, or that you can separate your positive cells from the autofluorescence?

@@chugcytometry3284 Separating positive cells from autofluorescence. There is positive signal in all serially-diluted antibody samples but no clear positive and negative populations (all tubes, including unstained, look like a lot of fluorescence spread).