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

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.