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.