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Flow cytometry whole blood samples: Red blood cell Lysis

Flow cytometry is often used for detection of various protein/antigens in blood samples, particularly for immunophenotyping. Analysis of the results is usually much simpler and more accurate when the red blood cells are not included in the sample. They need to be lysed.

Procedure

Red cells in whole blood samples for flow cytometry can be lysed using 0.1% Triton X-100 in PBS for 30 minutes (after brief fixation of whole blood with 2% or 4% formaldehyde). There are also several commercially available red blood cell lysing kits available (the manufacturer's kit protocol should be followed).

Research

Several detergents have been reported to affect detection of surface and internal antigens of blood cells. In the following reference, the impact of different detergents and detergent concentrations on RBC lysis and on WBC resolution using light scatter were investigated. Only three detergents, TX-100, NP-40, and Brij-58, showed significant RBC lysis after 2% formaldehyde fixation for 10 min at room temperature. These detergents had a deleterious effect on WBC light scatter at detergent concentrations above 0.2%, whereas concentrations below 0.1% resulted in incomplete RBC lysis. Thirty minutes of incubation at room temperature with 0.1% detergent was optimal. 2005 International Society for Analytical Cytology Cytometry Part A 67!:4-17 2005 Whole Blood Fixation and Permeabilization Protocol with Red Blood Cell Lysis for Flow Cytometry of Intracellular Phosphorylated Epitopes in Leukocyte Subpopulations.

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Red Blood Cell Lysis

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