Ductal and acinar cells from fresh tissue were isolated as previously described using HPx1 (acinar) and CD133 (ductal) markers (74 (link)).
To obtain ductal and de-differentiated acinar cells from in vitro cultures exocrine preparations were cultured as previously described (59 ,70 (link)) in Advanced RPMI 1640 medium containing 5% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin solution (Life Technologies) under 5% CO2 atmosphere at 37 °C. FITC-conjugated UEA-1 (Ulex Europeaus Agglutinin-1, Sigma, Overijse, Belgium) lectin labeling of acinar cells was performed according to Houbracken et al. and Baldan et al. (59 ,70 (link)). Exocrine cell fraction labeled with UEA-1 was kept in suspension culture. At day 4 of culture, cell clusters were dissociated following the protocol of Baldan et al. (59 ). Cells were stained with carbohydrate antigen 19.9 (mouse monoclonal anti-human CA19.9, Dako, Heverlee, Belgium). Alexa Fluor 647 anti-mouse (Jackson Laboratory, Westgrove, PA, USA) was used as secondary antibody. Analysis and cell sorting were performed on a BD FACSAria (BD Biosciences). Viable, single cells were gated based on forward and side scatter.
To obtain ductal and de-differentiated acinar cells from in vitro cultures exocrine preparations were cultured as previously described (59 ,70 (link)) in Advanced RPMI 1640 medium containing 5% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin solution (Life Technologies) under 5% CO2 atmosphere at 37 °C. FITC-conjugated UEA-1 (Ulex Europeaus Agglutinin-1, Sigma, Overijse, Belgium) lectin labeling of acinar cells was performed according to Houbracken et al. and Baldan et al. (59 ,70 (link)). Exocrine cell fraction labeled with UEA-1 was kept in suspension culture. At day 4 of culture, cell clusters were dissociated following the protocol of Baldan et al. (59 ). Cells were stained with carbohydrate antigen 19.9 (mouse monoclonal anti-human CA19.9, Dako, Heverlee, Belgium). Alexa Fluor 647 anti-mouse (Jackson Laboratory, Westgrove, PA, USA) was used as secondary antibody. Analysis and cell sorting were performed on a BD FACSAria (BD Biosciences). Viable, single cells were gated based on forward and side scatter.
