Dan Levy's Lab 2018
Elian Abayev
Apoptosis detection protocol using the Annexin-V and PI kit
Needed reagents:
MEBCYTO Apoptosis kit (MBL, Nagoya)
Propidium Iodide (PI), or DAPI instead
Annexin-V FITC
Binding buffer (of the kit or home-made)
*** When seeding cells, keep in mind that you'll need some cells for control- cell without stain, with FITC Annexin v and PI alone. Once forgotten, consider splitting your samples before staining.
Optional "home-made" binding buffer
100mM HEPES, 140mM NaCl and 25mM CaCl2, pH 7.4
Cell collection (for adherent cells)
Collect SUP- medium of your cells and centrifuge it (600g/5min), it is very important to keep these dead cells for your experiment!
Wash the wells once with PBSx1
Add warm trypsin
When cells begin to detach (varies between cells), take the SUP (medium) of the cells you centrifuged in the first point, using this medium collect the cells to the same tube.
Centrifuge again. In case you wish to split the cells, you may pipet the pellet with the newly harvested cells and transfer them to new tubes.
Discard SUP. Gently wash cell pellet with PBSx1.
Centrifuge again. Discard SUP.
*** In case your cells are extremely sensitive, you may skip points 6-7 and continue to the next step. Just make sure you removed the medium.
Staining
Following the manufacturers protocol- resuspend the cells with 85µl binding buffer (here is another step where you may split your samples)
Prepare mix of the staining solution- 1µl Annexin-V FITC and 0.5µl PI (this is enough for 500,000 cells) in 150µl of additional (to the previous 85µl) binding buffer. Count the wells you have (let's assume you have one 6-well plate, hence, take 150*7 binding buffer and 7µl Annexin-V and 3.5µl of PI)
For your control wells- do not make mix, since you'll be staining with each stain separately.
Keep tubes in dark at room temp.
Transfer your samples to a proper FACS plate\tube, and read your results!