ADM Assay for Pancreatic Duct Formation

The ADM assay was carried out as previously described^{7 (link)} with slight modifications. Briefly, dissociated primary pancreatic acinar cells were resuspended in DMEM with 1% FBS, 2.5 μM of dexamethasone, and 0.25 mg/mL trypsin inhibitor. This suspension was mixed with a solution containing rat tail collagen (Thermo Fisher Scientific), 10× DMEM, and 0.34 M NaOH. The mixture was poured into 6-well plates, and the collagen gels were allowed to solidify for 30 minutes. Then the enriched cell culture media were added on top of the gel. In certain experiments, the enriched medium was spiked with exogenous IL-6 (PeproTech, Cranbury, NJ) at 2 or 12 ng/mL. The lower concentration of IL-6 was comparable to the concentration of IL-6 detected in the conditioned medium of RAW264.7 cells stimulated with LPS (100 ng/mL) for 6 hours. Enriched media were changed the next day (day 1) and then every 2 days. The total number of ducts per well were counted manually on day 5 using 10× magnification.