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I am perfoming cell cycle measurements with primary cardiac fibroblasts. Recently, I have added an RNase treatment step (37°C, 30min) to my protocol, which now gives me much sharper peaks. However, I suddenly have two new problems, which somehow must have to be related to this RNase treatment: First, in the FL2-W/FL2-A plot, I observe two long, horizontal lines for the G1/G0 and G2/M populations (indicative of massive cell clustering?). Second, additional smaller peaks appear in the FL2-A histogram, as if there were cells containing 6n and 8n of chromosomes. The latter problem, I haven't found anywhere in literature. I have attached an example of the FL2-W/FL2-A plot.
These are the protocols, which I have used:
Without RNase treatment:
1) Trypsinise cells, wash them once in citrate buffer and fix them by adding 100% EtOH forcibly expelled form the pipet. Vortex shortly and store at -20°C (at least o/n)
2) Centrifugation, resuspension in citrate buffer
3) Addition of PI / Na2HPO4 buffer immediately before FACS analysis
With RNase treatment:
1) Heat inactivation of contaminating DNases in RNase solution (99°C, 5min) and storage in aliquots at -20°C
2) Steps 1+2 as described above
3) Addition RNase + PI / Na2HPO4 buffer 37°C, 30min
4) FACS analysis
Does anyone have a clue, what these problems could mean and why they appear only after prolonged incubation with the PI buffer or RNase?
Thx in advance for you hints!
