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…
開示
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 |
