Hi,
I've been performing a kill curve assay for puromycin with 293T cells. 5 days post puro addition, it appears that the vast majority of cells (90+%) had detached, forming very large 'mats' of floating cells and very few isolated cells remaining attached. Assuming the floating cells were dead, I added trypan blue to see if the remaining cells were dead or not.
The results were: most of the attached cells turned blue (dead), but the detached cells in general didn't stain. Given that most of the attached cells were dead, I feel like the floating cells are dead and didn't stain because they floated (incubated the dye with cells for 3+ hours), but apparently live 293T cells can be rounded and detached, so I'm not confident?
Would be grateful if anyone could provide some insight :)
Hello!
You said that you "incubated the dye [trypan blue] with cells for 3+ hours", right? I'm not an expert in 293T cells but your protocol sounds strange, because it usually takes a couple of minutes to stain the most cell types.
Could you please add more details? How exactly did you perform staining and observing? Did you mix an example of cell suspension with equal volume of trypan blue? Did you place a droplet of the mixture into hemocytometer chamber?
Hi,
3 hours of trypan blue incubation sounds strange to me.
Usually I take 10 µl cell suspension, mix it with 10 µl trypan blue and immediately analyze the cells (e.g. counting). Dead cells are easily distinguishable from living cells. Works for pretty much all cells I work with (iPSC, HEK, Fibroblast).
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