I am currently doing NK degranulation assays by first sorting NK cells as CD56+ cells via FACS Aria III with 85um Nozzle. I am currently getting nearly 30% of CD107a expression on sorted NK cells without PMA+Ionomycin. If I use PBMCs in the assay I get 10% CD107a expression before PMA+Ionomycin and 65% CD107a expression after PMA+Ionomycin. Does anyone had a similar experience with FACS sorted cells? If so what should I do because I need to sort NK subsets via FACS for my project.
Hi Özgür,
What % live cells do you get after sorting? Dead cells may stain positive for CD107a. A gentle alternative to FACS is a magnetic bead-based depletion where T cells, B cells, etc are removed, leaving purified NK cells that have never been labeled with antibodies. I prefer the Dynabeads Untouched Human NK Cells Kit.
Chris
Thank you for your answer. We use dead cell labeling after sorting and viability is
It may happen due to mechanical and electrical force applied on cells.
To avoid this you need to switch to MACS.
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