Ber ep4

Huh7 cells were trypsinized, washed, and resuspended in Hank’s balanced salt solution (Lonza, Basel, Switzerland) supplemented with 1% HEPES and 2% PBS. Cells were incubated with the fluorescein isothiocyanate-conjugated anti-EpCAM monoclonal antibody BER-EP4 (DAKO) on ice for 30 min prior to cell sorting using FACSAriaII (BD Biosciences, San Jose, CA, USA). Sorted cells were harvested on dishes and cultured overnight for PCR analysis.

Mononuclear cell fractions from bone marrow blood were centrifuged as cytospins (Cytospin Centrifuge, Hettich, Germany) using 5x10⁵ cells per spot and slide. Slides were air-dried and stored dry and tightly sealed at -20 °C until further use. Cells were stained after 5 minutes aceton fixation, either with the primary pan-cytokeratin antibody A45-B/B3 detecting CK8, CK18 and CK19 (AS Diagnostik, Germany) or anti-EpCAM antibody BER-EP4 (Dako, Hamburg) using the Dako REAL detection system (Dako, Hamburg, Germany). Cytospins were analysed with an ACIS (automated cellular imaging system; Chromavision medical systems, St. Juan Capistrano, CA, USA) followed by manual microscopy by an independent scientist. Only positive cells with distinct morphological signs of a tumor cell were counted as positive cells [18 (link)]. Detection of at least one positive tumor cell regarded this patient as a positive case.