Wed Oct 19, 2022 4:37 pm by mleipold
Hi Giorgio,
What CyTOF model are you using? The *_Processed part isn't common.
Can you describe your wash steps after surface antibody staining? And can you explain what your panels are in each of the 3 figures? My understanding is that:
"CD3 CD56" has only CD3 and CD56, and does not contain CD8 (or maybe CD8 in a non-Cadium channel)
"CD8(6) CD8(12)" contains CD8 but not CD3 (or maybe CD3 in a non-Cadmium channel)
"CD3 CD8(6)" contains both CD3 and CD8 in Cadmium channels.
Is that correct? If so, insufficient washing and/or Ab titer might explain part of the diagonal you're point out.
One way you might be able to check: Gate your CD3+ cells, then plot CD4 vs CD8. If you have ion-exchange/scrambling, then I would expect a decent amount of CD4+CD8+ since the CD4+ would be falsely CD8+ due to scrambling. If this is instead isotopic impurity spillover (with no scrambling), then I would expect the CD4+ to remain CD8- and have little or no CD4+CD8+.
Also, NK cells are CD3- but some are CD8+. If your signal is coming from isotopic impurity-driven spillover (rather than metal ion exchange/scrambling), then those CD8+ NK cells should still have some "CD3"/110Cd signal and the CD8- NK cells shouldn't.
Mike