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

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Erythrocyte isolation from whole blood

1. Add 500 µL of whole blood in acid citrate dextrose (ACD) (stored at 4 °C) to a microcentrifuge tube.
   NOTE: Whole blood was purchased in ACD. According to the company, 1.5 mL of ACD is added to 7 mL of whole blood (8.5 mL total volume).
2. Centrifuge the whole blood at 700 x g for 5 min at room temperature (RT) and remove the clear plasma and the thin buffy coat using a pipette to leave the red erythrocyte layer.
3. Prepare 1 L of Ringer solution containing 125 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 32 mM HEPES, 5 mM glucose, and 1 mM CaCl₂. Adjust the pH to 7.4 by adding 2 µL drops of 1.0 M NaOH. To prepare a glucose-free Ringer solution, follow the same protocol, but do not include glucose in the solution.
4. Wash the erythrocytes 2x in Ringer solution by suspending the cell pellet in 1.5 mL of Ringer solution, centrifuging at 700 x g for 5 min at RT, and removing the supernatant.
5. Make a 0.4% hematocrit by resuspending 40 µL of the erythrocyte pellet in 9,960 µL of glucose-free Ringer solution to reach a final volume of 10 mL.
   NOTE: Hematocrit is a term used to refer to the volume fraction of erythrocytes in suspension. A 0.4% hematocrit is a suspension containing 0.4% erythrocytes.
6. Incubate the cell suspension at 37 °C for 7 days.

2. Treatment of erythrocytes with ionomycin and measurement of hemolysis

1. Dissolve 1 mg of ionomycin calcium salt in 630 µL of dimethyl sulfoxide (DMSO) to reach a final concentration of 2 mM. Aliquot and store at -20 °C.
2. Take 1 mL of the 0.4% hematocrit from step 1.5 and add 0.5 µL of 2 mM ionomycin to reach a final concentration of 1 µM. Incubate for 2 h at 37 °C.
   1. Use 1 mL of the hematocrit with no ionomycin treatment as a negative control.
3. Centrifuge the ionomycin-treated and untreated hematocrits at 700 x g for 5 min at RT and remove their supernatants to leave the cell pellets at the bottom of the tubes.
4. Wash the cells 3x with Ringer solution by suspending the cell pellets in 1.5 mL of Ringer solution, centrifuging at 700 x g for 5 min at RT, and discarding the supernatants.
5. To measure hemolysis, add 1 mL of the untreated 0.4% hematocrit from step 1.5 to a microcentrifuge tube and incubate for 2 h at 37 °C as the negative control for hemolysis (0%).
6. Add 1 mL of the untreated 0.4% hematocrit from step 1.5 to a microcentrifuge tube and centrifuge at 700 x g for 5 min at RT. Remove the supernatant and add 1 mL of distilled water to the cell pellet and incubate for 2 h at 37 °C as the positive control for hemolysis (100%).
7. Add 1 mL of the ionomycin-treated 0.4% hematocrit from step 2.2 to a microcentrifuge tube.
8. Centrifuge the untreated cells, treated cells, and the cells in distilled water at 700 x g for 5 min at RT.
9. Take 200 µL of the supernatants and add to a 96-well plate.
10. Measure the absorbance at 541 nm using a microplate reader.
11. Calculate the hemolysis using Equation 1:
    %Hemolysis = (A_T – A₀)/(A₁₀₀ – A₀)*100
    where A₀ is the absorbance of erythrocytes in Ringer solution, A₁₀₀ is the absorbance of erythrocytes in water, and A_T is the absorbance of treated erythrocytes by ionomycin.