I need to do an flow cytometry experiment on SH-SY5Y to access the presence of some surface markers. Does anybody know at which time point the SH-SY5Y has more expression of extracellular markers, considering the cells viability? Can I just grown to range ~80% of confluence or more and do the staining?
I wanted to follow a staining protocol that I have used previously, but it requires collecting cells at 24, 48 and 72h, and at these time points there is a lot of cells in suspension and the cell viability is not suitable for the assay.
Hi Marília Sena ,
I am sure you already realized SH-SY5Y cells have mixed properties, adherence and suspension cells. Depends on your experiment, usually you can choose which types of cell growth properties you prefer. For my case, I usually grown adherence cells only and discard those suspension cells, leave more rooms for adherence cells to grow.
Back to your question at which time point does it express more extracellular marker, you need to optimize and examine at those time point suggested. Usually you need to wait up to 70-80% cells confluency. For each time point, the first day you seed the cells you seed at the same cell density and incubate for 24, 48 & 72 hours. And then do the staining after respective incubation time. Then flow cyto will you give results at which time point your cells achieve optimal expression. Then you can select the time point for subsequent analysis or further repetition.
Aimi Syamima Abdul Manap Thank you for your considerations! I'm goind to use T75 flasks and seed them for optimal growth and then do the staining. Since i'm only confirming the presence/absense of some receptors, I won't follow the time points for now. And I'm going to use both adherent and floating cells.
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