Detecting Apoptosis with Annexin V/PI - (Jul/18/2007 )

Hello everybody,

having a problem investigating apoptosis of Jurkat cells under the fluorescent microscope using an detection kit from Sigma Annexin V-FITC Apoptosis Detection Kit (APOAF).

I guess that the labelling procedure worked fine. However, the investigation under the fluorescent microscope (FM) doesn't give me any results.

I have a set of live jurkat cells, and apoptotic cells. Apoptosis was induced with UV irradiation for 30 sec. Additionally, to one set, I added a protein, and to another set the Buffer I have used.

After fixing, labelling, and mounting the coverslips onto the slides, I set up the FM with the slide with the live cells i.e. FM was set up so that you barely can see FITC and PI on the image. Mounting the slides with apoptotic cells, I was not able to see any difference i.e. no cells showed up for FITC and PI. However, by increasing the exposure time and the sensitivity, cells can be seen.

Any suggestions? Cells were incubated on coverslips, then fixed on the slips, followed by labelling the cells, at last mounted on the slides.

Thanks for your help.

hello,

I never fix the cells before staining with annexin V
even for storage, let the cells in mounting medium
do you use annexin buffer (with calcium etc...)?

Seb

Not really answering your question but a different way to approach detection of apoptosis- Do you have access to a flow cytometry machine? I recently treated PC12 cells to induce apoptosis and stained with Annexin V and 7AAD to detect apoptosis- worked beautifully, gives a percentage of cells that are apoptotic so no need to count cells manually .