Annexin V Binding Assay: A Fluorescence-Based Technique to Identify Apoptotic Erythrocytes via Phosphatidylserine Labeling

Abstract

Source: Bigdelou, P., et al. Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore. J. Vis. Exp. (2020)

In this video, we demonstrate the Annexin V binding assay to measure the degree of eryptosis, erythrocyte programmed cell death, using fluorescent Annexin V staining. Apoptotic erythrocytes demonstrate phosphatidylserine translocation from the inner to the outer leaflet of the cell membrane, causing the fluorophore-tagged Annexin V to bind to the translocated phosphatidylserine, thereby emitting a fluorescent signal.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Annexin-V binding assay

1. Dissolve 1 mg of ionomycin calcium salt in 630 µL of dimethyl sulfoxide (DMSO) to reach a final concentration of 2 mM. Aliquot and store at -20 °C.
2. Take 1 mL of the 0.4% hematocrit and add 0.5 µL of 2 mM ionomycin to reach a final concentration of 1 µM. Incubate for 2 h at 37 °C.
3. Centrifuge the ionomycin-treated and untreated hematocrits a....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Annexin V Alexa Fluor 488 - apoptosis kit	Fisher Scientific	A10788	Store at 4 °C
BD FACSAria II flow cytometer	BD Biosciences	643177
Ionomycin calcium salt	EMD Milipore Corp.	407952-1MG	Dissolve in DMSO to reach 2 mM. Store at -20 °C
Centrifuge	Millipore Sigma	M7157	Model Eppendorf 5415C
Disposable round bottom flow cytometry tube	VWR	VWRU47729-566
CaCl2	Fisher Scientific	C79-500
DPBS	VWR Life Science	SH30028.02
Microcentrifuge tube	Fisher Scientific	02-681-5
Whole blood in ACD	Zen-Bio		Store at 4 °C and warm to 37 °C prior to use