Novel Cell Imaging Techniques Show Induction of Apoptosis and Proliferation in Mesothelial Cells by Asbestos

• DOI: 10.1165/ajrcmb.17.3.2991
• Publication Date: 2013-04-04T02:26:09Z
• Authors: Jonathan L. Goldberg; Christine L. Zanella; Yvonne M. W. Janssen; Cynthia R. Timblin; Luis A. Jimenez; Pamela Vacek; Douglas J. Taatjes; Brooke T. Mossman
• Keywords: DAPI; Terminal deoxynucleotidyl transferase

We developed in situ dual-fluorescence detection techniques for measuring apoptosis and proliferation simultaneously single dishes of cells. The deoxyribonucleic acid (DNA)-specific labeling method, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end (TUNEL), first was used conjunction with a 4',6-diamidino-2-phenylindole (DAPI) counterstain to detect measure morphologic characteristics apoptotic rat pleural mesothelial (RPM) cells isolated from Fischer 344 rats exposed 300 microM hydrogen peroxide (H2O2). For this purpose, 100 TUNEL-positive nuclei were measured while being viewed DAPI counterstaining area, perimeter, longest diameter, average using imaging software an image-collection apparatus. then range concentrations crocidolite asbestos putative mitogenic agents. Exposure (5 microg/cm2) caused striking dose-dependent response at 24 h, 48 72 h. nonfibrous analogue riebeckite failed induce apoptosis. At tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml) increase nuclei. A second utilizing antibody 5'-bromodeoxyridine (BrdU) oxazole yellow homodimer (YOYO), showed occurring h In addition, increased numbers 12-O-tetradecanoylphorbol-13-acetate (TPA), TNF-alpha, epidermal growth factor (EGF) exhibited incorporation BrdU these time points, although total per unit area unchanged. Results indicate dynamic balance between DNA synthesis after exposure asbestos.