I am currently working on the T24 bladder cancer line and I am co-culturing for 72h with immunosuppressive myeloid cells generated in vitro. After 72h of co-culture, I want to isolate T24 cells by performing a CD45-/+ sorting.
I tried a cytometric sort using BD FACS Melody with a viability marker (Sytox Blue) and CD45-APC antibody. I never managed to recover any cells despite using a sorting buffer with 2–4 mM EDTA (PBS + BSA + EDTA), filtering at 30 µM before sorting, and working on ice throughout the experiment. My "event rate" is very labile (probably linked to the existence of cell aggregates) and the low number of cells recovered are dead.
I also tried a MACS sort with CD45 microbeads (Miltenyi) with the Automacs pro (Miltenyi). I sorted negatively on CD45 and recovered the CD45 fraction. This is slightly better, but my yield is still very low, with a large proportion of cells ending up in the CD45+ fraction.
Does anyone have experience in sorting this cell line (T24-bladder cancer)?
Thank you in advance for your suggestion.
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