As I understand, we aspirate medium, wash w PBS, aspirate, add trypsin and stop it with medium , centrifuge (I normally centrifuge 1000 rpm for 5 min , decant SN, resuspend pellet in medium and divide into groups to different stain, centrifuge again. My question is what speed this second centrifuge ?.
Hi Heba,
Normally I centrifuge (depeding of cell) in the same rotation like the first. I don't know what kind cell do you work, but I always keep the same rotation in both steps.
Well first of all I will never use Trypsin when I am going to do a cell membrane marker staining (e.g. Annexin V). Please use Trypsin free solution (EDTA/PBS). Centrifuge rotation could be used between 800-1200 rpm for 3 min at room temp.
Annexin V staining would take 30 min for 0.1 million cells and after that 2 washes of 1X PBS (1200 rpm for 3 min) and then counter stain for 20 min and 2 washes (1200 rpm for 3 min). Re-suspend in at least 300 micro lit Assay buffer and do Flow cytometry.
Good luck!!
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