Hello everyone,
I m looking for an Annexin V staining protocol for adherent cells without detaching cells from plate. As known, using trypsin to detach cells can increase considerably the number of false positive.
Could you please share a protocol?
Respectfully,
Hamadi
Hi Hamadi, how do you intend to analyse these cells then? With some experimentation you could probably use a confocal microscope to image Annexing V labelled cells but this is quite irregular. I would suggest you optimise your detachment process as publishing a FACS based Annexing V assay will be considerably easier. Maybe try diluting your trypsin, or substituting for a gentler protease such as accutase or collagenase. In my experience the number of positive cells in a negative control sample is rarely an issue.
Dear Hamadi,
I culture cells in wells attached to a microscope slide ( =chamber slides) that have the size of the microscope objective. This really works well.
1) Once you have done the treatment you want, and want to evaluate with Annexin V, wash the cells with PBS, twice.
2) Calculate the amount of Annexin V following the supplier's protocol (depends of the amount of cells).
3) Incubate for 10 min at RT the wells with Annexin V.
4) Look under microscope in a dark room. Beware, Annexin V does faint away after a few minutes.
Good luck!
Sandrine
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