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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.
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.
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 membrane1,2. Loss of asymmetry results in the translocation of phosphatidylserine (PS), a lipid normally localized in the inner leaflet3,4, to the cell outer leaflet, which signals to macrophages to phagocytose and remove defective erythrocytes5,6,7,8. 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 thalassemia9,10,11, 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 aminophospholipids13,14,15,16. 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 erythrocytes12,17,18,19,20,21,22,23,24,25,26. Ionomycin has both hydrophilic and hydrophobic groups, which are necessary to bind and capture Ca2+ ion, and transport it to the cytosolic space27,28,29. 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.
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
2. Treatment of erythrocytes with ionomycin and measurement of hemolysis
3. Annexin-V binding assay
4. Flow cytometry
5. Confocal microscopy
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 ...
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...
The authors have nothing to disclose.
This work was supported by NIH grant R15ES030140 and NSF grant CBET1903568. Financial support from the Russ College of Engineering and Technology and the Department of Chemical and Biomolecular Engineering at Ohio University is also acknowledged.
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 |
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