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

Overview

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 at 700 x g for 5 min at RT, and remove their supernatants to leave the cell pellets at the bottom of the tubes.
4. Wash the cells 3x with Ringer solution by suspending the cell pellets in 1.5 mL of Ringer solution, centrifuging at 700 x g for 5 min at RT, and discarding the supernatants.
5. Resuspend the ionomycin-treated and untreated erythrocyte cell pellets in 1 mL of 1x binding buffer.
6. Dilute 2 mL of the 5x annexin V binding buffer in 8 mL of phosphate-buffered saline (PBS) to obtain 1x binding buffer.
7. Take 235 μL of the cell suspensions in the binding buffer and add 15 μL of Annexin V-Alexa Flour 488 conjugate.
8. Incubate the cells at room temperature (RT) for 20 min in a dark place. Centrifuge at 700 x g for 5 min at RT and remove the supernatant.
9. Wash the cells 2x with 1x binding buffer, by suspending the cell pellet in 1.5 mL of the binding buffer, centrifuging at 700 x g for 5 min at RT, and removing the supernatant.
10. Resuspend the cell pellets in 250 μL of 1x binding buffer for flow cytometry measurements.

2. Flow cytometry

1. Transfer 200 μL of the annexin V-stained erythrocytes to 1 mL round bottom polystyrene tubes compatible with flow cytometry.
2. Login to the flow cytometry software and click on the new experiment button. Click on the new tube button. Select the global sheet and choose the apply analysis to measure the fluorescence intensity with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.
3. Set number of cells to 20,000 to be collected for fluorescence-activated cell sorting (FACS) analysis.
4. Select the desired tube and click on load button. Click on record button for forward scatter and side scatter measurements. Repeat for all samples.
5. Right-click on specimen button and click on apply batch analysis to generate the result file.
6. Right-click on specimen button and click on generate FSC files.

Materials

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