Encyclopedia of Experiments: Biological Techniques
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Hemolysis Assay - ConceptTranscript
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.
