Membrane cell permeabilization with saponin and multiparametric analysis by flow cytometry.
Saponin, a detergent like molecule, can permeabilize cell membranes without destroying them, and thus can be used for the detection of intracellular antigens on intact cells with a flow cytometer. First experiments were reported that demonstrated the detection of intracytoplasmic antigens such as intermediate filaments and CD3 in T acute lymphoblastic leukemia (ALL). Further experiments were also performed to prove that intranuclear structures were equally accessible: dyes such as propidium iodide (PI) and monoclonal antibodies (mAb) such as Ki67 could penetrate the nucleus and lead to the analysis of DNA content and to the discrimination between the different cell cycle phases (G0, G1, S, G2-M). This rapid and sensitive method retained sufficient integrity of cells being treated to enable differentiation of cell types on the basis of morphology. Furthermore, it did not alter membrane expression of most antigens. Therefore, it was of particular interest for multiparametric analysis, especially for simultaneous study of membrane and intracellular structures.
Jacob
M C
MC
Laboratoire d'Immunocytologie, Centre de Transfusion Sanguine de Grenoble BP 35, La Tronche, France.
Favre
M
M
Bensa
J C
JC
D000782:Aneuploidy; D015703:Antigens, CD / Q000032:analysis; D000951:Antigens, Neoplasm / Q000032:analysis; D000954:Antigens, Surface / Q000032:analysis; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002469:Cell Separation; D004273:DNA, Neoplasm / Q000032:analysis; D005434:Flow Cytometry; D006801:Humans; D019394:Ki-67 Antigen; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology; D008214:Lymphocytes / Q000187:drug effects / Q000276:immunology; D008223:Lymphoma / Q000276:immunology / Q000473:pathology; D009687:Nuclear Proteins / Q000032:analysis; D012503:Saponins / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000276:immunology
D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D004273:DNA, Neoplasm; D019394:Ki-67 Antigen; D009687:Nuclear Proteins; D012503:Saponins