Re: Does anyone experienced spillover or contamination like
Thanks for the pictures of your gating.
As I understand it, you do
1. Ungated-->Ir193 vs Event length to get Intact Singlets.
2. Intact Singlets-->CD45 vs Event length to get CD45+
3. CD45+--> CD4 vs CD8
Did you have CD66, CD14 or CD33, and CD3 in your CyTOF panel?
If so, I would advise revising your gating:
1. Ungated-->Ir193 vs Event length to get Intact Singlets.
2. Intact Singlets-->CD45 vs CD66 to get CD45+CD66-
3. CD45+CD66- ---> CD33 vs CD14 to get CD33-CD14-
4. CD33-CD14- ---> CD3 histogram or CD3 vs CD20 (or CD19) to get CD3+ (CD20-/CD19-)
5. CD3+ --> CD4 vs CD8
The reason for this is that it's possible that your CD3 depletion was not as successful as you would like. If so, then CD4+ and CD8+ cells would have made it through your purification.
Regarding MACS beads: while the beads themselves *are* iron oxide, iron is 54Fe-58Fe (92% 56Fe). Therefore, it won't be in the mass window of even a CyTOFv2, which is good, considering the amount of iron in your buffers, water, and cells.
In other words, it's the metal contaminants *in* the beads that are the issue, not the iron itself.
Mike