A simple method for the flow cytometric analysis of intracellular antigens in whole smooth muscle cells: quantification of cyclin-dependent kinase 2.
Flow cytometry has become a widely used technique for the quantitative analysis of antigens at the single cell level. In the past, several protocols have been published for the detection of cytoplasmic and nuclear antigens in various cell lines, especially blood cells or cells growing in fluid culture. The applicability of these protocols to cells growing in a monolayer, such as smooth muscle cells (SMC) is often restricted, although flow cytometry is of great interest in the fields of arteriosclerosis and cancer research. We here describe a simple and reproducible method for the flow cytometric analysis of intracellular antigens such as cyclin-dependent kinase 2 (Cdk2) in rat aortic SMC. The sensitivity of the method was analyzed under growth and growth-inhibitory conditions using lovastatin, a cholesterol-lowering compound with antiproliferative capacity. Various antigens (Ras-protein, protein kinase C-alpha (PKC-alpha), Ki-67/MIB-1) in rat and bovine SMC were detectable using this methodology which should have a wide range of applications.
Sindermann
J
J
Institute for Arteriosclerosis Research, University of Münster, Germany.
Weigel
K A
KA
Breithardt
G
G
D000818:Animals; D042846:CDC2-CDC28 Kinases; D002417:Cattle; D002453:Cell Cycle; D002467:Cell Nucleus / Q000201:enzymology; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000032:analysis*; D003593:Cytoplasm / Q000201:enzymology; D004247:DNA / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D015151:Immunoblotting; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D017346:Protein Serine-Threonine Kinases / Q000032:analysis*; D051381:Rats
D004247:DNA; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases