Amylase assay kit

Amylase activity was determined by colourimetric assay, using Abcam amylase assay kit, according to the manufacturer’s protocol (22 ). amylase assay kit detects the activity of α-amylase through a two-step reaction. The α-amylase cleaves the substrate ethylidiene-pNP-G7 to produce smaller fragments that are modified by α-glucosidase, causing the release of a chromophore that can be measured at A=405 nm. The assay can detect α-amylase activity as low as 0.2 mU.

The plasma was separated and stored at −80°C for ELISA assay. The α-amylase activity of plasma was assessed using the amylase assay kit from Abcam (Cambridge, UK) in accordance with the manufacturer's instructions. 25 μl of the plasma sample was diluted to 50 μl for each test, and the activity of α-amylase was recorded as mU/ml (nmol/min/ml). The plasma HMGB1 levels were detected by HMG1 / HMGB1 ELISA Kit obtained from LifeSpan BioSciences (Seattle, WA) according to the manufacturer's description.

To assess indices of tissue injury, blood was collected from mice 24 h following sham or CLP procedure via cardiac puncture using a heparin-coated syringe. Blood sample was centrifuged at 10,000 rpm, and supernatant (plasma) was collected and stored at -80°C. For tissue injury assays, plasma was analyzed using the following kits according to the manufacturer’s protocol: Urea Nitrogen (BUN) Colorimetric Detection Kit (Invitrogen, Carlsbad, CA, USA, cat# EIABUN), Creatine Kinase Activity Assay Kit (Sigma-Aldrich, St. Louis, MO, USA: cat# MAK116), Alanine Aminotransferase (ALT) Activity Assay Kit (Sigma-Aldrich: cat# MAK052), Aspartate Aminotransferase (AST) Activity Assay Kit (Sigma-Aldrich: cat# MAK055), Amylase Assay Kit (Colorimetric) (Abcam, Cambridge, MA, USA: cat# ab102523), and Bilirubin Assay Kit (Direct Colorimetric) (Abcam: cat# ab235627).

Pancreatitis was induced by cerulein injections as above. At 12 hours following the last injection, blood was collected from the caudal vena cava into BD Microtainer tubes (Thermo Fisher Scientific). Samples were centrifuged at 2,000g for 5 minutes at 4°C to obtain serum. An Amylase Assay Kit (Abcam) was used to determine serum amylase levels.

Pancreatitis was induced by cerulein injections as above. At 12 hours following the last injection, blood was collected from the caudal vena cava into BD Microtainer tubes (Thermo Fisher Scientific). Samples were centrifuged at 2,000g for 5 minutes at 4°C to obtain serum. An Amylase Assay Kit (Abcam) was used to determine serum amylase levels.