A rapid flow cytometric method for the detection of intracellular cyclooxygenases in human whole blood monocytes and a COX-2 inducible human cell line.
We have developed a flow cytometric method for the detection of intracellular cyclooxygenases (COX) in human whole blood monocytes and a COX-2 inducible human cell line. COX-2 is induced by endotoxin activation of whole blood monocytes or by the addition of fetal bovine serum (FBS) to a serum-deprived human fibroblastoid cell line, CCD-1070Sk. Cells are permeabilized with FACS Lysing Solution (FLS) containing saponin (Sap), stained intracellularly with COX-2 and COX-1 monoclonal antibodies (mAbs) and analyzed flow cytometrically. Intracellular COX-2 is specifically detected in endotoxin-stimulated CD14(+) monocytes in whole blood and in the inducible cell line. The specificity of COX-2 and COX-1 binding is demonstrated by competitive inhibition studies in cells and binding studies on protein-conjugated beads. In addition, a two-color reagent combination is described which simultaneously detects COX-2 and COX-1. We conclude that specific, intracellular COX-1 and COX-2 expression can be readily identified by flow cytometry in whole blood monocytes and cultured cells. The relative rapidity, ease of use and small sample volume required by this assay makes it a suitable methodology for studying COX expression in both preclinical and clinical research settings.
Ruitenberg
Joyce J
JJ
BD Biosciences, Immunocytometry Systems, 2350 Qume Drive, San Jose, CA 95131, USA.
Waters
Cory A
CA
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D003116:Color; D016895:Culture Media, Serum-Free; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D003593:Cytoplasm / Q000201:enzymology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007527:Isoenzymes / Q000032:analysis* / Q000096:biosynthesis* / Q000276:immunology; D008565:Membrane Proteins; D009000:Monocytes / Q000201:enzymology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000032:analysis* / Q000096:biosynthesis* / Q000276:immunology; D012503:Saponins / Q000494:pharmacology; D013997:Time Factors
D000911:Antibodies, Monoclonal; D016895:Culture Media, Serum-Free; D007527:Isoenzymes; D008565:Membrane Proteins; D012503:Saponins; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases