For cell cycle analysis I fixed my cells using 75% ethanol (1mL cold PBS + 3mL 100% EtOH (-20'C), dropwise), and stained them with Propidium Iodide (PI). It is supposed to be a homogeneous cell line.
After gating the singlets (lower left graph, FL-2A vs FL-2W) it still looks like I have 2 different cell populations when I visualize FL-2A vs FSC-H. Does anyone know whether this is the case (or that it looks like this is the case) or that this is due to an artefact (eg. fixation)? If the latter is the case, which population should I use for cell cycle analysis?
Hello,have you seen the cells under microscope?maybe you have two type of cells with different size.some times in cell lines cultures you have different types of cells but you see them same.
another point that sometimes in cell lines you have different DNA mass in your cells because of mutation or hyperploidy so you see separated cell cycles which have overlap in histogram
I agree with Jan, looking at FSC-W by FSC-H is the best way to look for singlets and doublets. It may be that your cells are aggregating and giving you two distinct populations. Also, I've found in the past that EtOH is a somewhat harsh way to fix cells and leads to permeabilization so this may also cause shifts in cell size. Fixing in 2% paraformaldehyde in PBS will fix cells without permeabilizing and may help in your analysis.
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