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Methods for simultaneous measurement of apoptosis and cell surface phenotype of epithelial cells in effusions by flow cytometry

Nature Protocols volume 3, pages 955–964 (2008)Cite this article

Abstract

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.

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Figure 1: Gating strategy for cell lines.
Figure 2: dUTP incorporation and cleaved caspase-3 and capase-8 expressions in cell lines.
Figure 3: Analysis of effusion specimens.
Figure 4: Degradation of PerCP conjugate by ethanol in the APO-DIRECT assay.

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Acknowledgements

This work was supported by the Norwegian Cancer Society, the Southern Norway Health Region Research Fund and the Research Fund at The Norwegian Radium Hospital.

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Authors and Affiliations

  1. Division of Pathology, Norwegian Radium Hospital, Rikshospitalet Medical Center, Ullernchaussen 70, Montebello, Oslo, N-0310, Norway

    Hiep Phuc Dong, Lilach Kleinberg, Ben Davidson & Björn Risberg

  2. Faculty Division Radiumhospitalet, The Medical Faculty, University of Oslo, Forskningsveien 3A, Oslo, N-0373, Norway

    Ben Davidson

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Correspondence to Björn Risberg.

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Dong, H., Kleinberg, L., Davidson, B. et al. Methods for simultaneous measurement of apoptosis and cell surface phenotype of epithelial cells in effusions by flow cytometry. Nat Protoc 3, 955–964 (2008). https://doi.org/10.1038/nprot.2008.77

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