Permeabilization and fixation conditions for intracellular flow cytometric detection of the T-cell receptor zeta chain and other intracellular proteins in lymphocyte subpopulations.
Expression of the T-cell receptor (TCR) zeta chain in normal individuals was studied by two-color flow cytometric analysis using digitonin-permeabilized human peripheral blood lymphocytes. Optimal detection of the TCR zeta chain involved fixation of cells in 0.25% paraformaldehyde for 2 min and permeabilization with 500 microg/ml of digitonin at 4 degrees C. Permeabilized lymphocytes and monocytes displayed decreased forward light scatter properties. Neutrophils did not survive this permeabilization/fixation/washing procedure. Permeabilization did not affect the ability of antibodies to CD3, CD4, CD8, CD16, and CD19 to detect CD antigens on lymphocytes, and the percentage of lymphocytes reacting with these antibodies was comparable between untreated and permeabilized cells. Staining of the TCR zeta chain was accomplished by incubating permeabilized cells with unconjugated antibody to the zeta chain, followed by an FITC-conjugated F(ab')2 goat anti-IgG. Following TCR zeta chain staining, cells were blocked with 25 microg/ml of mouse IgG for 20 min to saturate the goat anti-mouse antibody and then incubated with a phycoerythrin-conjugated mouse antibody to a variety of lymphocyte surface antigens. The TCR zeta chain was observed in lymphocytes displaying CD3, CD4, CD8, CD16, or TCRgammadelta markers. This permeabilization/fixation/washing procedure was also validated for detection of another intracellular T-cell protein known as the cytolytic granule-associated protein, recognized by the TIA-1 antibody. Thus the technique can be applied to detection of at least two different intracellular T-cell markers.
Department of Immunohaematology, St. Michael's Hospital, Toronto, Ontario, Canada.
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Lazarus
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Ellis
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Blanchette
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Freedman
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Sheng-Tanner
D000911:Antibodies, Monoclonal / Q000737:chemistry; D015703:Antigens, CD / Q000276:immunology; D002463:Cell Membrane Permeability; D004072:Digitonin; D005404:Fixatives; D005434:Flow Cytometry / Q000379:methods*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008027:Light; D008565:Membrane Proteins / Q000032:analysis*; D011948:Receptors, Antigen, T-Cell / Q000032:analysis*; D012542:Scattering, Radiation; D016176:T-Lymphocyte Subsets / Q000737:chemistry* / Q000378:metabolism; D016707:Tissue Fixation / Q000379:methods*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D005404:Fixatives; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; C054979:antigen T cell receptor, zeta chain; D004072:Digitonin