CyTOF to study aged stem cells
I’m experiencing some troubles with the Cell Prep and DNA labeling of aged stem cells for CyTOF2 and I hope that you could give me some suggestions with your expertise. I am just starting out using mass cytometry. Right now I’m testing optimal cell Prep conditions to get single cell suspensions. I use DNA intercalator(Ir-191/193) only to look at my cell populations on CyTOF2 during the optimization phase. I have observed that approximately 50% of the events in the fcs file are of low event length and lower Ir -191 intensity than the small percentage of cells labeled with the DNA Ir-191/193 intercalator. I tried different Fix/Perm buffers and different dilutions of DNA intercalator. It looks like The Fix/Perm buffer from ebioscience (used for Foxp3 staining) resulted in more effective labeling of single intact cells than Max par Fix/perm buffer. I also tested 3 dilutions of DNA intercalator, and it seems that 1:2000 dilution works better than 1:1000 and 1:500. There is another problem with these senescent cells. After detaching, 40% cells are dead based on try pan blue staining and create lots of cell debris. I wonder if this can cause lots of non-specific bindings. These aged cells have both DNA condensation and decondensation and I wonder if this kind of cell phenotype may be causing problems with the DNA intercalator? Please review the attached screenshots (dot plots, rain plots and cell suspension images) for more details. The dot plots are DNA (Ir-191) vs. EQ4 beads (Ce140) and DNA (Ir-191) vs. event_length . I’d really appreciate if you could give me some insight on optimizing cell prep, DNA intercalation and/or suggesting alternative strategies to label aged stem cells with a metal-tagged reagent that will aide in cell singlet ID using CyTOF2.