A new membrane permeabilization method for the detection of intracellular antigens by flow cytometry.
This article describes a new cell membrane permeabilization method for the detection of intracellular antigens by immunofluorescence staining and flow cytometry. The number of cells remained unaltered and no cell aggregation or loss of intracellular antigenicity was observed after this permeabilization treatment. A mixed leukocyte population from human peripheral blood was used in this study and the leukocytes were fixed and permeabilized, which permitted monoclonal anti-vimentin antibodies to reach intracellular antigens. The stabilization of cell membranes and preservation of intracellular antigenicity was achieved with paraformaldehyde fixation. This pretreatment prevents cell destruction and subsequent treatment with the detergent n-octyl-beta-D-glucopyranoside results in permeabilization of the cell membrane. The procedure does not alter the expression of cell surface antigens, which is of importance if phenotypic characterization of intracellularly stained cells is to be undertaken. Furthermore, this simple, rapid and reproducible technique makes it possible to detect and distinguish between different human peripheral blood leukocytes without prior purification steps. The leukocyte subpopulations remain well-separated and easily detectable by flow cytometry.
Halldén
G
G
Department of Clinical Immunology, Karolinska Hospital, Stockholm, Sweden.
Andersson
U
U
Hed
J
J
Johansson
S G
SG
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis*; D002463:Cell Membrane Permeability; D005434:Flow Cytometry; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D005557:Formaldehyde; D005960:Glucosides; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000276:immunology*; D007962:Leukocytes / Q000187:drug effects / Q000276:immunology*; D008027:Light; D011108:Polymers; D011309:Preservation, Biological; D012542:Scattering, Radiation; D013194:Staining and Labeling; D014746:Vimentin / Q000276:immunology
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D005452:Fluoresceins; D005960:Glucosides; D011108:Polymers; D014746:Vimentin; D005557:Formaldehyde; C018619:octyl-beta-D-glucoside; C024098:6-carboxyfluorescein; C003043:paraform