Methods for simultaneous measurement of apoptosis and cell surface phenotype of epithelial cells in effusions by flow cytometry.
We describe here a protocol for the detection of epithelial cells in effusions combined with quantification of apoptosis by flow cytometry (FCM). The procedure described consists of the following stages: culturing and induction of apoptosis by staurosporine in control ovarian carcinoma cell lines (SKOV-3 and OVCAR-8); preparation of effusion specimens and cell lines for staining; staining of cancer cells in effusions and cell lines for cell surface markers (Ber-EP4, EpCAM and CD45) and intracellular/nuclear markers of apoptosis (cleaved caspase-3 and caspase-8, and incorporated deoxyuridine triphosphates); and FCM analysis of stained cell lines and effusions. This protocol identifies a specific cell population in cytologically heterogeneous clinical specimens and applies two methods to measure different aspects of apoptosis in the cell population of interest. The cleaved caspase and deoxyuridine triphosphate incorporation FCM assays are run in parallel and require (including sample preparation, staining, instrument adjustment and data acquisition) 8 h. The culturing of cell lines requires 2-3 days and induction of apoptosis requires 16 h.
Division of Pathology, Norwegian Radium Hospital, Rikshospitalet Medical Center, Ullernchaussen 70, Montebello, N-0310 Oslo, Norway.
Hiep Phuc
HP
Dong
Lilach
L
Kleinberg
Ben
B
Davidson
Björn
B
Risberg
D017209:Apoptosis; D001202:Ascitic Fluid / Q000378:metabolism / Q000473:pathology; D001826:Body Fluids / Q000166:cytology*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D010051:Ovarian Neoplasms / Q000378:metabolism / Q000473:pathology; D010490:Pericardial Effusion / Q000378:metabolism / Q000473:pathology; D010641:Phenotype; D010996:Pleural Effusion / Q000378:metabolism / Q000473:pathology; D013194:Staining and Labeling
D003856:Deoxyuracil Nucleotides; D020169:Caspases