Hemolysis Assay: An In Vitro Method to Measure Calcium Ionophore-Induced Lysis in Human Erythrocytes
Published: April 30, 2023
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 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 add…
Disclosures
The authors have nothing to disclose.
Materials
| 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 |
Tags
Cite This Article
Hemolysis Assay: An In Vitro Method to Measure Calcium Ionophore-Induced Lysis in Human Erythrocytes. J. Vis. Exp. (Pending Publication), e21179, doi: (2023).