I am going to do DNA content analysis on HeLa cells and I searched for relevant protocols. Some of them suggest do PI staining and RNase A together, while others stated it was better to do RNase A first and then PI staining. I have no idea what the differences are between these two versions of protocols.
Dear Siyuan, I always use RNase A and PI at the same time (is in the same buffer), I incubate the cells for min. 15 min before they are subjected to flow cytometry. Works always pretty fine :)
Dear Wolfgang, thanks for your answer. Another quick question: how would you quench the reaction of PI after 15min incubation? Just by standing the tubes on ice? Someone suggest wash with 1*PBS, I doubt if it is necessary, because I am afraid of the cell loss during the wash process.
Dear all,
After the incubation with PI/RNase, do we need to spin down the cells and remove the PI/RNase buffer before analyse it using Flow cytometer?
Thanks.
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