AnnV-PI-7AAD

Annexin V (AnnV) & Propidium Iodide (PI) or 7-aminoactinomycin D (7AAD) Staining

Last updated: 10/7/13 By: Tim Starr

Overview

AnnV binds phosphatidylserine, which is normally in the cell membrane facing the cytoplasm. As a cell undergoes apoptosis, the phosphatidylserines flip and face externally and will bind to AnnV. Therefore, AnnV is a marker of apoptosis.

PI and 7AAD only enter dead cells and bind to the DNA, thus they are markers of necrotic cell death.

Before staining with Annv, PI or 7AAD, you can stain cells with antibodies that won't fluoresce in the same channels as AnnV, PI or 7AAD. Double check to make sure your cells do or do not already contain a fluorescent gene such as GFP, YFP, RFP, etc.

AnnV comes conjugated to a fluorescent molecule such as FITC-conjugated and APC-conjugated AnnV.

The emission maximum for DNA-bound Propidium Iodide is approximately 615–620 nm. When excited by a 488 nm laser, PI can therefore be detected in both the PE-Cy5 (FL-3) as well as the PE channel (FL-2). This means you really can't use a PE- or a PE-Cy5- conjugated antibody with Propidium Iodide.

7AAD/DNA complexes excited by the 488 nm laser (Actual excitation is 546 nmand have an emission maxima of 647 nm (FL-3).

Protocol

• Generally use 5e5 cells per sample, can range from 2e5 to 1e6. Can stain in tubes or 96 well-plates
• Spin plates or tubes and wash 2 times in 200 µl ice cold PBS
• Spin plates or tubes and resuspend in 100 µl Annexin V Binding Buffer that contains 5 µl of Annexin V and either 5 µl of Propidium Iodide or 1 µl of 7AAD (1 mg/ml in DMSO).
• Incubate at room temp for 15 minutes in the dark
• Add 100 µl of additional binding buffer to each sample (don’t spin) and FACS as soon as possible (the protocol says within 1 hour)

7AAD

Invitrogen/Molecular Probes 7-Aminoactinomycin D (7-AAD) Catalog # A1310. 1 mg. Dissolve in DMSO at 1 mg/ml. Use 1 µl of 7AAD per 1 ml of Annexin V buffer containing 1e6 cells.

Annexin V - APC

Thermo/Fisher Scientific Annexin V APC, Catalog #BDB550474. 500 µl solution. Use 5 µl per sample.

Annexin V Buffer

Mix ingredients and adjust pH to 7.4. Store at 4ºC