Sep 29, 1989

A rapid sample preparation technique for flow cytometric analysis of immunofluorescence allowing absolute enumeration of cell subpopulations

Journal of Immunological Methods
L W TerstappenM R Loken

Abstract

A simple and rapid method was developed for immunofluorescence measurements of cells by flow cytometry which does not require washing procedures, permitting absolute enumeration of cell subpopulations. Peripheral blood cells were labeled with fluorescein and phycoerythrin conjugated monoclonal antibodies and the nucleic acid stain LDS-751. Distilled water was added following incubation to induce erythrocyte lysis by hypotonic shock. After lysis for 30 s the tonicity of the sample was increased followed by measurement on the flow cytometer. The leukocyte populations were clearly resolved in the correlation of forward and orthogonal light scattering. The immunofluorescence resolution of the labeled leukocytes was equivalent to NH4Cl and a commercial lysing preparation. Absolute number of leukocytes and percentage of leukocyte subpopulations determined with this procedure correlated well with the results obtained with a clinical hematology analyzer. Cell recovery and preservation of cellular characteristics of three different procedures for lysing the human erythrocytes were compared. The LDS-751 permitted the discrimination of intact cells from residual erythrocyte ghosts, platelets and damaged nucleated cells. A considerable los...Continue Reading

Mentioned in this Paper

Pathologic Cytolysis
Monoclonal Antibodies
Monoclonal antibodies, antineoplastic
Flow Cytometry
Immunofluorescence Assay
T6 Antigens
CD19 Antigens
Distilled water
White Blood Cell Count Procedure
Fluorescein

About this Paper

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