Fluorescent plate reader

HCASMC were loaded with Fura 2-AM (2 µM, Teflabs, USA) for 30 min at 37 °C in medium. Cells were then incubated in Krebs buffer and Fura-2AM fluorescence (excitation 340 and 380 nm, emission 520 nm) measured using a fluorescent plate reader (BMG Labtech Clariostar, Germany). Intracellular [Ca²⁺] was calculated using the Grynkiewicz formula^{57 (link)}.

After sacrifice of the mice, blood was allowed for natural clotting for 30 minutes and followed by centrifugation at 1,500 g × 10 minutes to collect serum. Pancreas and lung samples were also harvested and snap frozen for future use. A proportion of pancreas was also fixed by 10% formalin overnight before being subjected to H&E staining (5 μm thick per slide).
Serum amylase was tested in Clinical Biochemistry Department in Royal Liverpool University Hospital using a kinetic method. Serum IL-6 was measured by ELISA according to manufacturer's instruction (R&D, Abingdon, UK). Pancreatic trypsin activity was determined by established protocol using trypsin peptide substrate Boc-Gln-Ala-Arg-MCA (Peptide, Osaka, Japan) on a fluorescent plate reader (BMG Labtech, UK). Pancreatic and lung myeloperoxidase activity, pancreatic trypsin activity, and pancreatic histopathology were analysed by methods previously reported [23 (link)]. All histopathological scoring was undertaken in a double-blinded manner by independent assessors.