Krb buffer

For the Glucose-Stimulated Insulin Secretion (GSIS) assay, insulin secretion was measured by incubating 15 IPCs. First, IPCs were washed carefully with PBS and then incubated in Kreps Ringer Bicarbonate Buffer (Table 3; KRB buffer, Sigma-Aldrich, St. Louis, MO, USA), followed by incubation for 1 h in KRB buffer with 2.8 mM glucose (low glucose) or 17.5 mM glucose (high glucose). Insulin levels were measured in the supernatant after 1 h. Insulin secretion was determined using the human insulin ELISA Kit (Cat# ab278123; Abcam, Cambridge, MA, USA).

Generation of PDOs, culture, and drug studies were performed as previously described [17 (link)]. Briefly, ultrasound-guided PDAC biopsies were collected and placed onto a 60 mm Petri dish on ice in KRB buffer (Sigma-Aldrich, K4002, St. Louis, MO, USA) and minced into small pieces. Then, the chopped tissue was treated with 2.5 mg/mL collagenase type IV (Sigma-Aldrich, C4-BIOC) and penicillin/streptomycin (Gibco, 15140122, Grand Island, NY, USA) in KRB solution. The mixture was placed into a water bath at 37 °C for 5 min under agitation to digest the extracellular matrix. The extracted cells were collected by filtration in a 100 µm strainer and centrifugated at 1500 rpm for 5 min. To remove red blood cells in the cell pellet, 3 mL of RBC lysis buffer (Invitrogen, 00-4333-57, Carlsbad, CA, USA) was added for 3 min and deactivated with FBS (Gibco, 16000044). Finally, the extracted PDAC cells were seeded onto Matrigel-coated (Corning, 356255, Corning, NY, USA) 12-well plates in organoid media and cultured for 1 week until organoid formation. For subculture, the Matrigel and organoids were broken down using a gentle cell dissociation reagent, and the organoids were freshly embedded with 50% Matrigel-50% organoid media in a well plate. For chemotherapy, organoids were used within four passages.