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Best kit for apoptosis detections

flow cytometry apoptosis annexin V

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3 replies to this topic

#1 Elioelio

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Posted 26 June 2013 - 06:16 AM

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

#2 Curtis

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Posted 27 June 2013 - 04:34 AM

It could be your fixing. How do you fix?

#3 Elioelio

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Posted 28 June 2013 - 02:17 AM

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.

#4 Curtis

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Posted 28 June 2013 - 09:24 AM

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.





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