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

During certain pathological conditions, erythrocytes or red blood cells undergo cell shrinkage accompanied by translocation of membrane phospholipid - phosphatidylserine - from inner to the outer membrane leaflet, leading to programmed cell death, eryptosis, triggering macrophage-mediated cell phagocytosis.

To measure ionophore-induced eryptosis in isolated human erythrocyte population via Annexin V binding assay, begin with erythrocytes suspended in a glucose-free buffer.

Glucose depletion induces cellular stress, activating cytosolic protein kinase C, which subsequently activates calcium channels and causes calcium ion influx. Further, potassium channels get activated, causing potassium efflux and cell shrinkage.

Next, add calcium ionophore that forms a stable complex with extracellular calcium ions. The complex facilitates cytosolic entry of calcium ions across the cell membrane, further increasing intracellular calcium concentration. This, in turn, activates scramblase - a bidirectional phospholipid transporter - which aids in translocation of phosphatidylserine from the inner to the outer cell membrane leaflet, reducing membrane asymmetry and inducing eryptosis.

Centrifuge the ionophore-treated erythrocytes. Resuspend the cells in a calcium-containing binding buffer. Add fluorescent dye-conjugated Annexin V solution.

The calcium-dependent phospholipid-binding protein, Annexin V, having high phosphatidylserine affinity binds to the exposed phosphatidylserine residues on the outer cell membrane leaflet.

Using fluorescent-activated cell sorting, separate the erythrocyte population based on the Annexin V fluorescence intensity. Eryptotic erythrocytes exhibit higher Annexin V fluorescence intensity than healthy erythrocytes.

Dilute 2 milliliters of the 5X Annexin V binding buffer in 8 milliliters of PBS to obtain 1X binding buffer. Resuspend the ionomycin-treated and untreated cell pellets in 1 milliliter of the 1X binding buffer.

Take 235 microliters of the cell suspensions in the binding buffer, and add 15 microliters of Annexin V-Alexa Fluor 488 conjugate in a microcentrifuge tube. Incubate the cells at room temperature for 20 minutes in a dark place.

Centrifuge at 700 x g for 5 minutes at room temperature. Remove the supernatant. Wash the cells twice with 1X binding buffer by suspending the cell pellet in 1.5 milliliters of the binding buffer, and centrifuging at 700 x g for 5 minutes at room temperature.

Remove the supernatant and resuspend the cell pellets in 250 microliters of 1X binding buffer for flow cytometry measurements. Now, transfer 200 microliters of the Annexin V-stained erythrocytes to 1-milliliter round-bottom polystyrene tubes compatible with flow cytometry.

Log in 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 nanometers and an emission wavelength of 530 nanometers.

Set number of cells to 20,000 to be collected for fluorescence-activated cell sorting analysis. Select the desired tube and click on the 'Load' button. Click on 'Record' button for forward scatter and side scatter measurements. Repeat for all samples.

Right-click on the 'Specimen' button and click on 'Apply Batch Analysis' to generate the result file. Right-click on 'Specimen' button and click on 'Generate FSC Files.'