I've been trying to study cell cycle using PI (propidium iodide + Triton-X + sodium citrate)b staining solution.
I trypsinize my cells (A431: skin carcinoma), fix them using 100% methanol and store in 4 degree Celsius. I then wash the cells once with cold PBS and re-suspend them in the staining solution. Till this point it is a single cell suspension after vortexing well.
I leave the samples overnight in 4 degree Celsius and acquire using low cytometer the next day. But by this time the cells form clumps. These clumps are not broken down by vortexing or even by pipetting.
I've tried vortexing the sample again and again right before acquiring the cells. I've used freshly made PBS and PI staining solution. I've maintained adding cold solutions since every step after trypsinization. I've shifted to using methanol instead of ethanol for fixation.
I'm still unable to solve for the clump formation.
Sudeep Kumar Maurya I normally add the stain and then keep them at 4 degrees overnight. I have also tried with staining for 30 minutes at room temperature and acquiring it the same day, still the clumping issue persists. This clumping issue is something I'm facing with only A431 cells and not the other cell lines (WI38, A549, LN229)
Niharika Nema Hello did you find a solution to your problem? I am currently encoutering the same issue with Hela cells.
Chaima Fadhloun Hello.
So I stopped fixing my cells. I trypsinize, wash once with PBS and then add the PI-tritonX-Sodium citrate stain. I leave the cells in 4 degree Celsius overnight, and acquire it the next morning. Just before acquiring I vortex the samples, and use a cell filter.
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