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.
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.
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