After processing testicular tissue and isolating haploid germ cells, I fix them in 70% ethanol and store at 4 degree C. I usually perform flow cytometry cell cycle analysis 2-3 months after sample collection. Before using flow cytometry, I add Propidium Iodide and RNAse 1 and incubate for 30 minutes.
As you can see, the graph shows I have more cell debris than my haploid cells, my cells of interest. I can't use this graph and this is a waste of my precious samples.
Can someone please help me out? How do I remove the cell debris from my ethanol-fixed cell suspension to get a clear flow cytometry graph?
You can do a gentle centrifugation to pellet your cells and aspirate the debris-containing supernatant.
You should also be able to exclude cell debris with more careful gating during the flow run. Be sure you are basing your total counts for the run on the actual cells and not just every event that the cytometer detects.
John Hardy Lockhart Thank you. I centrifuge the cell suspension at 100x g, then remove the ethanol and resuspend the pellet in PBS before adding my fluorescent dye. Gating during flow cytometry is challenging, as most of my suspension contains cell debris, which is evident in the graph.
I don't want to use dead cell removal kit as I have a lot of samples and that might get expensive.
I've tried modifying my protocol but it hasn't resulted in anything.
You can increase the centrifuge speed to 300 × g; it may help you to decrease the debris and improve the gating during flow cytometry.
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