Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore

Summary

A protocol for the induction of eryptosis, programmed cell death in erythrocytes, using the calcium ionophore, ionomycin, is provided. Successful eryptosis is evaluated by monitoring the localization phosphatidylserine in the membrane outer leaflet. Factors affecting the success of the protocol have been examined and optimal conditions provided.

Abstract

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic cells is the loss of compositional asymmetry of the cell membrane, leading to the translocation of phosphatidylserine to the membrane outer leaflet. This process is triggered by increased intracellular concentration of Ca²⁺, which activates scramblase, an enzyme that facilitates bidirectional movement of phospholipids between membrane leaflets. Given the importance of eryptosis in various diseased conditions, there have been efforts to induce eryptosis in vitro. Such efforts have generally relied on the calcium ionophore, ionomycin, to enhance intracellular Ca²⁺ concentration and induce eryptosis. However, many discrepancies have been reported in the literature regarding the procedure for inducing eryptosis using ionomycin. Herein, we report a step-by-step protocol for ionomycin-induced eryptosis in human erythrocytes. We focus on important steps in the procedure including the ionophore concentration, incubation time, and glucose depletion, and provide representative result. This protocol can be used to reproducibly induce eryptosis in the laboratory.

Introduction

Programmed cell death in erythrocytes, also known as eryptosis, is common in many clinical conditions and hematological disorders. Eryptosis is associated with cell shrinkage and the loss of phospholipid asymmetry in the cell plasma membrane^1,2. Loss of asymmetry results in the translocation of phosphatidylserine (PS), a lipid normally localized in the inner leaflet^3,4, to the cell outer leaflet, which signals to macrophages to phagocytose and remove defective erythrocytes^5,6,7,8. At the end of the normal life span of erythrocytes, removal of eryptotic cells by macrophages ensures the balance of erythrocytes in circulation. However, in diseased conditions, such as sickle cell disease and thalassemia^9,10,11, enhanced eryptosis may result in severe anemia². Due to its importance in hematological diseases, there is significant interest in examining the factors inducing or inhibiting eryptosis and the molecular mechanisms underlying this process.

The plasma membrane of healthy erythrocytes is asymmetric, with different phospholipids localizing at the outer and inner leaflets. Membrane asymmetry is primarily regulated by the action of membrane enzymes. Aminophospholipid translocase facilitates the transport of aminophospholipids, PS and phosphatidylethanolamine (PE), by directing these lipids to the cell inner leaflet. On the other hand, floppase transports the choline containing phospholipids, phosphatidylcholine (PC) and sphingomyelin (SM), from the inner to the outer leaflet of the cell membrane¹². However, unlike healthy cells, the membrane of eryptotic erythrocytes is scrambled. This is due to the action of a third enzyme, scramblase, which disrupts phospholipid asymmetry by facilitating the bidirectional transport of aminophospholipids^13,14,15,16. Scramblase is activated by elevated intracellular levels of Ca²⁺. Therefore, calcium ionophores, which facilitate the transport of Ca²⁺ across the cell membrane¹², are efficient inducers of eryptosis.

Ionomycin, a calcium ionophore, has been widely used to induce eryptosis in erythrocytes^{12,17,18,19,20,21,22,23,24,25,26}. Ionomycin has both hydrophilic and hydrophobic groups, which are necessary to bind and capture Ca²⁺ ion, and transport it to the cytosolic space^27,28,29. This leads to the activation of scramblase and translocation of PS to the outer leaflet, which can be easily detected using annexin-V, a cellular protein with a high affinity to PS¹². Although triggering eryptosis by ionomycin is commonly reported, there is considerable method discrepancy in the literature (Table 1). The population of erythrocytes undergoing eryptosis depends on different factors such as ionophore concentration, treatment time with ionophore, and the sugar content of extracellular environment (glucose depletion activates cation channels and facilitates the entry of Ca²⁺ into the cytosolic space)^30,31. However, there is little consistency in these factors in the literature, making it difficult to perform eryptosis reproducibly in vitro.

In this protocol, we present a step-by-step procedure to induce eryptosis in human erythrocytes. Factors affecting successful eryptosis including Ca²⁺ concentration, ionophore concentration, treatment time, and pre-incubation in glucose-depleted buffer are examined and optimal values are reported. This procedure demonstrates that pre-incubation of erythrocytes in a glucose-free buffer significantly increases the percentage of eryptosis compared to glucose-containing buffer. This protocol can be used in the laboratory to produce eryptotic erythrocytes for various applications.

Protocol

All human blood samples used in the protocol described below were purchased as de-identified samples. No human subjects were directly involved or recruited for this study. The guidelines of the Declaration of Helsinki should be used when research involves human subjects.

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 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 1³²:
    
    %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.

3. Annexin-V binding assay

1. Dilute 2 mL of the 5x annexin V binding buffer in 8 mL of phosphate-buffered saline (PBS) to obtain 1x binding buffer.
2. Resuspend the ionomycin-treated and untreated cell pellets from step 2.4 in 1 mL of 1x binding buffer.
3. Take 235 µL of the cell suspensions in the binding buffer and add 15 µL of Annexin V-Alexa Flour 488 conjugate.
4. Incubate the cells at RT for 20 min in a dark place. Centrifuge at 700 x g for 5 min at RT and remove the supernatant.
5. Wash the cells 2x with 1x binding buffer, by suspending the cell pellet in 1.5 mL of the binding buffer, centrifuging at 700 x g for 5 min at RT and removing the supernatant.
6. Resuspend the cell pellets in 250 µL of 1x binding buffer for flow cytometry measurements.

4. Flow cytometry

1. Transfer 200 µL of the annexin-V stained erythrocytes to 1 mL round bottom polystyrene tubes compatible with flow cytometry.
2. Login 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 nm and an emission wavelength of 530 nm.
3. Set number of cells to 20,000 to be collected for fluorescence-activated cell sorting (FACS) analysis.
4. Select the desired tube and click on load button. Click on record button for forward scatter and side scatter measurements. Repeat for all samples.
5. Right click on specimen button and click on apply batch analysis to generate the result file.
6. Right click on specimen button and click on generate FSC files.
7. Add the flow cytometry data (FSC files) into the workplace of flow cytometry software.
8. Analyze the control data by selecting the cell population of interest and adding statistics for eryptosis value.
   1. Double click on control and select histogram versus fluorescence intensity.
   2. Click on gate button to draw a gate on the histogram which indicates the percentage of eryptosis.
9. Apply the same statistics for all other experimental tubes to obtain the eryptosis values. Right click on control and select copy analysis to group.
10. After properly gating all samples, transfer the analyzed data by dragging and dropping them into the layout editor.
    1. Overlay the analyzed data with control in layout editor.
    2. Set the desired histograms and intensities by changing the x and y axis of the overlaid graphs.
    3. Export image files by clicking on export button and save the graphs in desired location.

5. Confocal microscopy

1. Transfer 5 µL of annexin-V-stained cells on a microscope slide and cover it with a cover slip. Keep in a dark place to prevent photobleaching.
2. Use Argon laser of the confocal fluorescence microscope to observe the cells excited at 488 nm with desired magnifications.
   NOTE: A confocal microscope is not necessarily needed and any microscope with fluorescence capabilities can be used to obtain fluorescence images that demonstrate annexin-V binding.
3. Obtain fluorescence images of the control (non-treated cells) and treated cells.
   NOTE: Non-treated cells are expected to show very weak fluorescence signals, whereas treated cells are expected to show bright green fluorescence on their membranes.

Results

Optimization of ionomycin concentration

While ionomycin is required to induce eryptosis, increased ionomycin concentrations can lead to hemolysis (i.e. lysis of erythrocytes and release of hemoglobin), which needs to be avoided. Treatment of erythrocytes with 1 µM ionomycin in Ringer solution for 2 h is enough to induce eryptosis, as evidenced by successful labeling with annexin-V Alexa Flour 488 conjugate and q...

Discussion

The goal of this procedure is to provide optimal values for ionophore concentration, treatment time, and extracellular glucose concentration, which are important factors in ensuring successful induction of eryptosis. A critical step in the protocol is the depletion of extracellular glucose, which, despite its importance, has not been sufficiently emphasized in the literature. The sugar content in normal Ringer solution (5 mM) has an inhibitory effect on eryptosis. Glucose depletion in the extracellular environment induce...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well plate	Fisher Scientific	12-565-331
Annexin V Alexa Fluor 488 - apoptosis kit	Fisher Scientific	A10788	Store at 4 °C
BD FACSAria II flow cytometer	BD Biosciences	643177
CaCl2	Fisher Scientific	C79-500
Centrifuge	Millipore Sigma	M7157	Model Eppendorf 5415C
Confocal fluorescence microscopy	Zeiss, LSM Tek Thornwood		Model LSM 510, Argon laser excited at 488 nm for taking images
Cover glasses circles	Fisher Scientific	12-545-100
Disposable round bottom flow cytometry tube	VWR	VWRU47729-566
DMSO	Sigma-Aldrich	472301-100ML
DPBS	VWR Life Science	SH30028.02
Glucose monohydrate	Sigma-Aldrich	Y0001745
HEPES Buffer (1 M)	Fisher Scientific	50-751-7290	Store at 4 °C
Ionomycin calcium salt	EMD Milipore Corp.	407952-1MG	Dissolve in DMSO to reach 2 mM. Store at -20 °C
KCl	Fisher Scientific	P330-500
MgSO4	Fisher Scientific	M65-500
Microcentrifuge tube	Fisher Scientific	02-681-5
NaCl	Fisher Scientific	S271-500
Plain glass microscope slides	Fisher Scientific	12-544-4
Synergy HFM microplate reader	BioTek
Whole blood in ACD	Zen-Bio		Store at 4 °C and warm to 37 °C prior to use