Collagenase type xi from clostridium histolyticum

InSolution™ Staurosporine was from Calbiochem/EMD Millipore. Dimethyl Sulfoxide (DMSO), Thapsigargin (Tg), Palmitic Acid (PA), Propidium Iodide (PI), Tetramethylrhodamine Ethyl Ester Perchlorate (TMRE), Collagenase Type XI from clostridium histolyticum, corn oil and Tamoxifen (TM; Cat# T5648) were from Sigma-Aldrich (St. Louis, MO, USA). Fura-2/AM and Hoechst 33342 were from Invitrogen/Life Technologies (Burlington, ON, CA).

Cancer cells were labeled with CellTrackerTM Green 5-chloromethyl fluorescein diacetate (CMFDA). PCOs and CIPCOs were cultured for 4 days, followed by treatment with gemcitabine (Yuhan, Seoul, Korea) with or without 100 ng/mL collagenase (type XI from Clostridium histolyticum, Sigma-Aldrich) for 72 h. After that, PCOs and CIPCOs were treated with propidium iodide (PI; Sigma-Aldrich), and the PI-positive area was calculated to estimate the cancer cell death in the multicellular organoid model. Fluorescence images were acquired on a Lionheart FX Automated Microscope (Biotek, Winooski, VT, USA). After 72 h of drug treatment, green and red fluorescence was detected. Z-stack images were obtained by scanning an organoid every 10 μm from the top of the organoid with a total scan range of 100 μm in depth. A plane approximately halfway through the z-stack image was chosen for quantification, and the fluorescence intensities were plotted against the radius. Area analysis was processed using Gen5 Image Prime 3.08 (Biotek).