? Do you normally get 3 distinct subpopulations (indicated as R2-green , R3-blue and R4-red as in A. of the attached slide), or one uniform live population in the FSC/SSC as in B. of the attached slide? (I tried 2 different settings in SZ95 control cells stained with Nile Red).
R2 would correspond to SZ95 cells with increased size and increased granularity, R3 would correspond to large cells with less granularity and R4 to small sized cells with low granularity. And also, in order to assess Nile Red staining what gating strategy would you employ? For instance would you gate separately on each subpopulation and ask for FL1 or altogether?(We have a Partec FACS machine, equipped with a 488nm laser only, therefore we can detect Nile Red on the FL1 channel only and therefore can assess triglycerides and not polar lipids).
I have performed FACS analysis of SZ95 cells stained with CD44 variant and EpCAM, and remember that SZ95 cells are different from other cell lines due to the morphology. So, you have to check the gating of FSC and SSC.
Ref; Mammary epithelial stratification occurs through symmetry breaking vertical divisions of luminal cells (Biochem Biophys Res Commun. 2013 Sep 6;438(4):640-6.)
I would suggest to ensure first that these are single cells and not two cells stuck together, if you can, to run it on a cytometer that can get you FSC-H, FSC-W, FSC-A, SSC-H, SSC-W and SSC-A it would help loads on determining what those populations are morphologically. They look distinct however it is better to be safe than sorry. You may want to adjust your voltages some to seperate out the popultions a little more when you are sure. If they are distinct populations the best read out would be to gate them individually like you did and then look at FL1. You can always reflect those values back on the population in whole too! good luck
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