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

Abstract

Source: Bigdelou, P. et al., Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore. J. Vis. Exp. (2020).

This video describes an in vitro assay to determine calcium-ionophore-induced hemolysis or lysis of red blood cells in a human sample. The assay measures the percentage of hemolysis by quantifying hemoglobin released from ruptured erythrocytes.

Protocol

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 remov....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well plate	Fisher Scientific	12-565-331
CaCl2	Fisher Scientific	C79-500
Centrifuge	Millipore Sigma	M7157	Model Eppendorf 5415C
DMSO	Sigma-Aldrich	472301-100ML
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
Synergy HFM microplate reader	BioTek
Whole blood in ACD	Zen-Bio		Store at 4 °C and warm to 37 °C prior to use