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2 flasks in complete medium (RPMI 1640 supplemented with 10% fetal calf serum and glutamine). After overnight adhesion, unattached cells were discarded, whereas thyrocytes were removed from the flasks with 0.05% (w/v) trypsin. These cell preparations underwent a second digestion with dispase to obtain single-cell suspensions. Thyrocytes were then labeled with human serum containing anti-TPO, followed by labeling with fluorescein isothiocyanate-coupled goat anti-human Ig. Cells were then incubated for 10 min with 6% normal mouse serum before staining with phycoerithrin-conjugated UB2 mAb. Relative fluorescent intensities of individual cells were analyzed with a FACScan flow cytometer (Becton Dickinson).
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14
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1842318919
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note
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Fas-or CK-labeled cryostat thyroid sections (5 μm) were fixed in paraformaldehyde, permeabilized with 0.1% Triton X-100, and labeled by an in situ apoptosis detection kit (in situ cell death detection, alkaline phosphatase; Boehringer GmbH, Mannheim, Germany). Detection of labeled ends was done with anti-fluorescein, Fab fragment, conjugated with alkaline phosphatase. 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) (Dakopatts) was used as substrate. Sections were then counterstained with hematoxylin.
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Sections were allowed to equilibrate to room temperature and were exposed to hydrogen peroxide methanol solution for 5 min. Then slides were rinsed with distilled water and placed for 5 min in a tris buffer before staining with peroxidase-antiperoxidase. Control sections were exposed with control purified rabbit IgG. Anti-FasL (C-20, rabbit polyclonal IgG) was from Santa Cruz Biotechnology (Santa Cruz, CA). For flow cytometric analysis, freshly purified thyrocytes were treated with C-20 or control rabbit IgG, followed by phycoerithrin-conjugated goat anti-rabbit IgG (Sigma), as secondary reagent.
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+ cells were then removed after 30 min binding to sheep anti-mouse IgG-coupled beads (Dynal, Wirral Merseyside, UK) and magnetic depletion.
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23
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data not shown
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C. Giordano et al., data not shown.
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1842358543
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note
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Supported by Associazone Italiana Ricerca sul Cancro, Telethon, Consiglio Nazionale delle Ricerche, and the European Commission. We thank P. Bazan and G. Di Pace (Department of Surgery, University of Palermo) for surgical specimens and Genzyme Diagnostic (Cambridge, UK) for IL-1.
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