Cell detachment for flow cytometry expts - (Jan/04/2006 )

Hi All,

I am having some problems trypsinizing one of my cell lines (T47D) in the prep for some flow cytometry experiments. I find that if I treat them for 2 min at 37 C, I get a beautiful cell cycle plot, but about half of my cells remain stuck to the plate. To get all the cells to detach (even with fairly vigorous aspiration) I need to treat for 10 min at 37 C, but long before that I get a population of (presumably) sheared cells, showing less than one unit of DNA. Worse, I can't really gate this population out, since it is right on top of my normal cells in the FSC : SSC plot.

Any ideas? Would a non-enzymatic detachment reagent work better? Or maybe I should manually scrape the plate? I look forward to any suggestions.

Regards,
Chemist_guy

In addition to 2-5 minutes of tryplExpress (invitrogen), I manually scrape my epithelial cells with my pipette tip. Alternatively, 30 min of 0.02% EDTA in Hanks Balanced Salt Solution plus a little scraping works too.

Some of my cells look crappy on my FSC/SSC plot, but only a tiny amount of the total population, and very easily gateable.

Thanks Finnbar, but I'm afraid that won't do it. Even pipetting the cells up and down a few times causes two much <G1 stuff. Any other thoughts?

rinse your cell with filtered PBS (to get rid of FCS), then add trypsin.

Yup. Already do that.

Increase the volume of Trypsin to cover the cell.