Hemolysis Assay: An In Vitro Method to Measure Calcium Ionophore-Induced Lysis in Human Erythrocytes

Hemolysis is the rupturing of erythrocytes to release their cellular contents into the surrounding space.

To assay the hemolysis, begin with a tube containing erythrocytes in glucose-free, electrolytes-containing, isotonic solution relative to body fluids. This solution keeps erythrocytes intact, and the absence of glucose induces cellular stress. This activates the cationic channels allowing the entry of calcium ions into the cell's cytosolic space.

Add a solution containing an optimum calcium-ionophore concentration that binds the calcium ions and facilitates their transport into the cytosol, causing an overload of calcium ions inside erythrocytes. 

This influx activates scramblase, a protein that translocates the phospholipids between the plasma membrane's lipid monolayers, disrupting its asymmetry. This phenomenon also translocates phosphatidylserine, an inner monolayer phospholipid, to the outer monolayer, an indicator of eryptosis - programmed death of erythrocytes.

An increase in cytosolic calcium ions activate potassium channels and drives potassium ions out of the cell. An osmotic imbalance leads to water loss and shrinkage of the erythrocyte. A longer exposure to calcium ionophores induces hemolysis, causing hemoglobin leakage into the solution.  

Centrifuge RBC suspension and measure the absorbance of the supernatant to quantitate the amount of hemoglobin released from the hemolysed erythrocytes.