I stained HepG2 cells with propidium iodide (PI), calcein AM, and Hoechst. Dead cells were treated with PFA and Triton-X. The staining concentration and time were fine, but PI stained most of the cytoplasm in almost all cells, while Hoechst stained only the nuclei normally, indicating that the nuclei should be intact. I'm wondering if anyone has encountered this situation before and would appreciate any advice.
Hi Fang Song
If I got it wright you fixed the cells and then stained them with PI. If so, this is the problem. When you fix cell, PI would stain the cytoplasm.
Mohamed Khashan
Thank you so much for your answer. Indeed, I fix the cells with PFA before treating them with Triton-X. However, if I don't fix them, won't the dead cells be washed away? May I respectfully ask for your advice on how to avoid this situation?
Thank you very much.
Fang Song
If you wash them gently, no detachment would be observed e.g: add 300 ul of binding buffer to a well of 24-well plate, swirl gently then aspirate the buffer and go on.
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