Hi there:
I am working on 10 colour FACS in PBMC. For compensation reason, i need to induced PBMC to die, then it can stained by
LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit staining. To my knowledge, most people try: 1) 65 degree heat induce cells death;
2) PMA+Ionomycine culture; 3) DNA damaging chemicals, such as camptothecin to kill cells. Don't know which one is the best. Do you have any experience to share? Your comments may save a lot of my time, efforts and money. Any information is welcome.
Regards
Shiqiu
I initially used to heat kill at 65 degrees and stain with ebio live dead stain, which was good, but time consuming. Nowadays specific compensation beads are available. kindly check those. one such is arc amine reactive compensation bead.
Thanks for your response.
Julieta: I plan to stain surface marker+live/dead cell marker on live cells; then fix with PFA and do Perm, stain intracellular cytokine. Out of curiosity, why you are interested in fixative? Are you worrying PFA affect dead cell marker staining?
Thanks Dinesh, 65 degree heat can kill cells, mix intact cells would be good for live/dead cell marker staining. Just as you said, time and efforts. Thanks for letting me know the dead cell staining beads, which looks a good alternative for my auqa beads.
Regards
Yes Shiqiu Xiong i forgot to mention that the heat killed and intact live cells are mixed 1:1 ratio before staining with live dead stain.
If you need a positive control, just to compensate, you can induce death by PFA (mix 1:1 with live) and then stain with live/dead cell marker.
The easiest thing would be to fix and permeabilize your cells and then add your Live Dead stain. All the cells would be stained with Live/dead marker. Mix it 1:1 with unstained cells post staining. You will get good separation and it is less cumbersome.
I usually put around 10^6 cells in a vial and incubate at 95 degrees Celsius in a hot plate for 4-5 min. Then, after cooled down, mix those cells with equal amount of not-heated cells. Then do the Aqua staining. Usually I get beautiful positive and negative peaks.
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