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

Published: April 30, 2023

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 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. Take 1 mL of the 0.4% hematocrit and add 0.5 µL of 2 mM ionomyc…

Divulgaciones

The authors have nothing to disclose.

Materials

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

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Annexin V Binding Assay: A Fluorescence-Based Technique to Identify Apoptotic Erythrocytes via Phosphatidylserine Labeling. J. Vis. Exp. (Pending Publication), e21180, doi: (2023).

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