Expression data from of FACS separated acinar and duct cell at day 4 of suspension cultured human pancreatic exocrine cells

Donor pancreata were obtained from the Beta Cell Bank of the JDRF Centre for Beta Cell Therapy in Diabetes (Brussels, Belgium), from Pancreatic Islet Processing (ECIT center) of Diabetes Research Institute at the IRCCS San Raffaele Scientific Institute (Milan, Italy) and from the DRWF Human Islet Isolation Facility (Oxford, England). Full written consent for use of donor material for research was obtained according to Belgian, Italian and English laws. This project was approved by the Medical Ethical Committee of all institutions. Cells were cultured in 3D suspension culture for four days in Advanced RPMI supplemented with 5% FBS. We used microarray to determine differential expression of genes to human panreatic acinar and duct ells at day 4 of suspension culture At day 4, human exocrine cell clusters were dissociated using Neurocult™ enzymatic dissociation kit for adult CNS tissue (Stemcell™ Technologies), incubated for 5 minutes at 37°C while resuspending constantly. Cells were resuspended in medium, filtered over a 40μm filter, spun down, and resuspended in 3% FBS buffer. Cell yield was measured through the Countess automated cell counter (ThermoFisher Scientific). Cells were incubated with mouse monoclonal anti-human carbohydrate antigen 19.9 antibody (anti-CA19.9, Dako, Heverlee, Belgium; 2 μl per million cells in 200 μl) or isotype control (IgG1 kappa, Abcam, Cambridge, MA, USA; 2 μl per million cells in 200 μl) for 15 minutes at 4 °C. Cells were washed with FBS buffer (PBS + 3% FBS) and incubated for 15 minutes at 4 °C with secondary antibody Alexa fluor 647 anti-mouse (Jackson Laboratory, Westgrove, PA, USA; 2 μl per million cells in 200 μl). Analysis and cell sorting was performed on a BD FACSAria (BD Biosciences, Erembodegem, Belgium). Viable, single cells were gated based on forward and side scatter. After sort, cells were immediately collected in RLT buffer (Qiagen, Germantown, MD 20874, USA) and kept on ice. RNA extraction was immediately performed after sorting.