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,

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....

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 .......

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

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