Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore

There have been many efforts to induce eryptosis using ionomycin. However, the exact procedure for this process is not clearly outlined in the literature. We provide a step-by-step procedure to induce eryptosis using ionomycin.

The main advantage of this procedure is to induce eryptosis in human erythrocytes in a reliable and reproducible manner. To begin, add 500 microliters of cold whole blood in acid citrate dextrose to a microcentrifuge tube. Centrifuge the whole blood at 700 times g for five minutes at room temperature and remove the clear plasma and the thin buffy coat using a pipette to leave the red erythrocyte layer.

Prepare one liter of Ringer solution containing 125 millimolar sodium chloride, five millimolar potassium chloride, one millimolar magnesium sulfate, 32 millimolar HEPES, five millimolar glucose, and one millimolar calcium chloride. Adjust the pH to 7.4 by adding two microliter drops of one molar sodium hydroxide. To prepare glucose-free Ringer solution, follow the same protocol but do not include glucose in the solution.

Wash the erythrocytes twice in Ringer solution by suspending the cell pellet in 1.5 milliliters of Ringer solution, centrifuging at 700 times g for five minutes at room temperature and removing the supernatant. Then make a 0.4%hematocrit by resuspending 40 microliters of the erythrocyte pellet in 9, 960 microliters of glucose-free Ringer solution to reach a final volume of 10 milliliters. Incubate the cell suspension at 37 degrees Celsius for seven days.

Pre-incubation of erythrocytes in a glucose-free buffer significantly triggers eryptosis. High eryptosis was obtained after seven days of pre-incubation in the sugar-free buffer. Dissolve one milligram of ionomycin calcium salt in 630 microliters of DMSO to reach a final concentration of two millimolar.

Aliquot in 20 milliliters and store at minus 20 degrees Celsius. Take one milliliter of the prepared 0.4%hematocrit and add 0.5 microliters of two millimolar ionomycin to reach a final concentration of one micromolar. Incubate for two hours at 37 degrees Celsius.

Use one milliliter of the hematocrit with no ionomycin treatment as a negative control. Centrifuge the ionomycin treated and untreated hematocrits at 700 times g for five 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 solution by suspending the cell pellets in 1.5 milliliters of Ringer solution, centrifuging at 700 times g for five minutes at room temperature and discarding the supernatants.

To measure hemolysis, add one milliliter of the untreated 0.4%hematocrit to a microcentrifuge tube and incubate for two hours at 37 degrees Celsius as the negative control of 0%hemolysis. To prepare the positive control of 100%hemolysis, add one milliliter of the untreated 0.4%hematocrit to a microcentrifuge tube and centrifuge at 700 times g for five minutes at room temperature. Remove the supernatant and add one milliliter of distilled water to the cell pellet to resuspend and incubate for two hours at 37 degrees Celsius.

Next, add one 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 five minutes at room temperature. Transfer 200 microliters of the supernatants into each well of a 96-well plate.

Measure the absorbance at 541 nanometers using a micro plate reader. Calculate the hemolysis using the equation where A0, A100, and AT are the absorbance of erythrocytes in Ringer solution in water and the absorbance of treated erthyrocytes by ionomycin. Dilute two milliliters of the 5X Annexin-V binding buffer in eight milliliters of PBS to obtain 1X binding buffer.

Resuspend the ionomycin-treated and untreated cell pellets in one milliliter of the 1X binding buffer. Take 235 microliters of the cell suspensions in the binding buffer and add 15 microliters of Annexin-V Alexa Fluor 488 conjugate in a microcentrifuge tube. Incubate the cells at room temperature for 20 minutes in a dark place.

Centrifuge at 700 times g for five minutes at room temperature. Remove the supernatant. Wash the cells twice with 1X binding buffer by suspending the cell pellet in 1.5 milliliters of the binding buffer and centrifuging at 700 times g for five minutes at room temperature.

Remove the supernatant and resuspend the cell pellets in 250 microliters of 1X binding buffer for flow cytometry measurement. Now transfer 200 microliters of the Annexin-V stained erythrocytes to one milliliter round bottom polystyrene tubes compatible with flow cytometry. Log in to the flow cytometry software and click on the new experiment button.

Click on the new tube button. Select the global sheet and choose the apply analysis to measure the fluorescence intensity with an excitation wavelength of 488 nanometers and an emission wavelength of 530 nanometers. Set number of cells to 20, 000 to be collected for fluorescence activated cell sorting analysis.

Select the desired tube and click on the load button. Click on record button for forward scatter and side scatter measurement. Repeat for all samples.

Right click on the specimen button and click on apply batch analysis to generate the result file. Right click on specimen button and click on generate FSC files. In this protocol, treatment of erythrocytes with one micromolar ionomycin and Ringer solution for two hours was enough to induce eryptosis as evidenced by successful labeling with Annexin-V Fluor 488 conjugate and quantification by FACS analysis.

Higher concentrations of ionomycin at five and 10 micromolar resulted in a slight increase in eryptosis. However, such concentrations also enhanced hemolysis. Incubation of erythrocytes in ionomycin and Ringer solution for as little as 30 minutes was enough to induce eryptosis.

Increased incubation time increased the level of eryptosis for up to two hours. However, further incubation time resulted in a slight decrease in the level of eryptosis. The higher value of hemolysis after 180 minutes explains the reduction in eryptosis after the same amount of incubation as less viable cells existed upon 180 minutes of treatment with ionomycin.

Erythrocytes with ionomycin treatment showed a bright fluorescence signal to the binding of Annexin-V to phosphatidyl serine in the outer leaflet. In contrast, cells with no treatment showed a very weak fluorescence signal indicating very low eryptosis. Pre-incubation in a glucose-free buffer is an important trigger for induction of eryptosis.

Since eryptosis is observed in a wide range of diseases, the experiments following this protocol are field specific. In our lab, we are interested in examining the effects of eryptosis on membrane biophysical properties and membrane interactions with nanomaterials. Eryptosis is associated with a large number of diseases including diabetes, sickle cell anemia, and thalassemia.

Having a reproducible protocol for inducing eryptosis can help researchers in any of these fields to investigate further scientific questions specific to each field.