Best kit for apoptosis detections - (Jun/26/2013 )
I've been using an annexin V/PI kit to detect apoptosis in DLD1 and HT29 cells. However, I have read that annexin V may not be specific to apoptotic cells as the phosphotidylserine can also be seen on ruptured cells. Indeed, when I put a high concentration of carbohydrate on my cells (which I thought would induce necrosis) I saw an increase in Annexin V stained cells.
It would be good to have a kit which had Annexin V and another stain to show mitochondrial membrane potential, condensed chromatin or caspase activation, or some other marker of apoptosis. There are so many out there - I wondered if anyone could help me choose the best one?
Another thing - my untreated control cells show a high percentage of dead cells (mainly AnnexinV and PI positive. Some annexinV only. I wondered if there could be something innately wrong with my cell line?
Thanks for any help
It could be your fixing. How do you fix?
I don't fix the cells for Annexin V, it's done on live cells.
I wash cells with PBS, trypsinise, pellet 200g 5 mins, wash with cell staining buffer, pellet again, resuspend in Annexin V binding buffer with Annexin V-FITC and PI.
I don't remember how I stained for annexn v, but for PI I fixed with ice cold ethanol and kept in -20C before staining and loaded on the machine not more than 5 min post staining. If I left more than 30 min all cells were stained.