This article discusses the process of hemolysis, which involves the rupture of erythrocytes and the release of hemoglobin into the surrounding solution. The study outlines a method to quantify hemolysis by measuring hemoglobin absorbance after treating erythrocytes with calcium ionophores.
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.
Dissolve 1 milligram of ionomycin-calcium salt in 630 microliters of DMSO to reach a final concentration of 2 millimolar. Aliquot in 20 microliters and store at minus 20 degrees Celsius.
Take 1 milliliter of the prepared 0.4% hematocrit, and add 0.5 microliters of 2 millimolar ionomycin to reach a final concentration of 1 micromolar. Incubate for 2 hours at 37 degrees Celsius. Use 1 milliliter of the hematocrit with no ionomycin treatment as a negative control. Centrifuge the ionomycin-treated and untreated hematocrit at 700 times g for 5 minutes at room temperature, and remove their supernatants, to leave the cell pellets at the bottom of the tubes.
Wash the cells three times with Ringer's solution, by suspending the cell pellets in 1.5 milliliters of Ringer's solution, centrifuging at 700 times g for 5 minutes at room temperature, and discarding the supernatants.
To measure hemolysis, add 1 milliliter of the untreated 0.4% hematocrit to a microcentrifuge tube, and incubate for 2 hours at 37 degrees Celsius as the negative control at 0% of hemolysis.
To prepare the positive control of 100% hemolysis, add 1 milliliter of the untreated 0.4% hematocrit to a microcentrifuge tube, and centrifuge at 700 times g for 5 minutes at room temperature. Remove the supernatant and add 1 milliliter of distilled water to the cell pellet to resuspend, and incubate for 2 hours at 37 degrees Celsius.
Next, add 1 milliliter of the ionomycin-treated 0.4% hematocrit to a microcentrifuge tube. Centrifuge the untreated cells, treated cells, and the cells in distilled water at 700 times g for 5 minutes at room temperature.
Transfer 200 microliters of the supernatants into each wall of a 96-well plate. Measured the absorbance at 541 nanometers using a microplate reader.
Calculate the hemolysis using the equation where A0, A100, and AT are the absorbance of erythrocytes in Ringer's solution, in water, and the absorbance of treated erythrocytes by ionomycin.
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