Apoptosis Detection with AnnexinV and PI
From Heit Lab Wiki
- Binding Buffer
- AnnexinV-flourophore conjugate (e.g. ThermoFisher AnnexinV-488)
- Propridium Iodide
Prepare binding buffer, for 100 mL:
- 238 mg HEPES (10 mM)
- 818 mg NaCl (140 mM)
- 37 mg CaCl2.2H2O (2.5 mM CaCl)
100 mL ddH2O pH to 7.4
Suspension Cells, or for FACS
- To 100 ul of binding buffer add 1 ug/ml (1 ul of a 100X solution) propridium iodide + 5 ul of AnnexinV conjugate
- Pellet 1x105 cells (suspend by trypsinization or scriping first, if required), wash 1x with binding buffer, then resuspend in the PI/AnnexinV buffer
- Incubate in the dark at room temperature for 15 min
- Add an additional 400 ul of ice-cold binding buffer, and store samples on-ice for imaging. Image or FACS analyzie as soon as possible.
- Grow cells on coverslips and treat as per your protocol
- Prepare 100 ul of labeling buffer/coverslip: 0.5 ug/ml (0.5 ul of a 100X solution) propridium iodide + 2.5 ul of AnnexinV conjugate
- Using the hanging drop method, place the live and unfixed cells on a droplet of labeling buffer, at room temperature, for 15 min in the dark.
- If imaging live, mount coverslip in spaceship and image immeidatly.
- If fixing/mounting the sample:
- During the incubation, prepare 4% PFA in binding buffer (not in PBS)
- Wash coverslip 1x with binding buffer, then add 4% PFA solution. Incubate 20 min at room temperature.
- Wash coverslip 1x with binding buffer, then mount slide and image as per normal