Apoptosis Detection with AnnexinV and PI

Reagents

• Binding Buffer
• AnnexinV-flourophore conjugate (e.g. ThermoFisher AnnexinV-488)
• Propridium Iodide

Protocol

Binding Buffer

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

1. To 100 ul of binding buffer add 1 ug/ml (1 ul of a 100X solution) propridium iodide + 5 ul of AnnexinV conjugate
2. Pellet 1x10⁵ cells (suspend by trypsinization or scriping first, if required), wash 1x with binding buffer, then resuspend in the PI/AnnexinV buffer
3. Incubate in the dark at room temperature for 15 min
4. 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.

For Microscopy

1. Grow cells on coverslips and treat as per your protocol
2. 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
3. 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:
   1. During the incubation, prepare 4% PFA in binding buffer (not in PBS)
   2. Wash coverslip 1x with binding buffer, then add 4% PFA solution. Incubate 20 min at room temperature.
   3. Wash coverslip 1x with binding buffer, then mount slide and image as per normal