Hello, I'll perform flow cytometry (Caspase-1, Annexin-V, PI assay) in differentiated macrophages (from THP-1 monocytes to macrophages using PMA). I usually detach the cells using TryPLE and wait up to 40 min for a complete detachment without the need to use scrapers. Does Tryple Affects Flow cytometry? I mean, does this substance need to be removed? Thank you all for your comments and suggestions.
That's a long time for trypsinization. You want to be as gentle as possible to avoid chopping up your antigens.
I only go 5-10 minutes with tryple for epithelial cancer lines. As soon as you see a slightly opaque surface, you should be able to swirl the media or use a pipettor to dislodge the cells.
When you add complete media, trypsin will go off target to the FBS, and your cells are more protected. Then centrifuge and resuspend.
I also think that the time of trypsinization is too long. Both trypsin and scraper damage the cell membrane, while the error can be reduced by a blank control.
we have found tryple select to be toxic to macrophages with far fewer numbers recovered for flow anlysis in comparison with trypsin. We are not sure what the cause is. They seem to just lyse either in the tryple or in the centrifugation steps. We also found macrophages don't lift like other cells, but can be "loosened" by tryple or trypsin (depending on whether endpoint included analysis of surface markers). ie they aren't all off the plastic, but can be gently squirted off using a pipette after 10 minutes.
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