Human insulin elisa kit

Insulin concentration (ng/mL or pM) was measured using a rat insulin ultrasensitive ELISA kit (Cat No. 62IN2PEG, Cisbio, Cambridge, England) or human insulin ELISA Kit (Cat No. 90095, CrystalChem, IL, USA), respectively, according to the manufacturer’s protocol.

Human insulin (27.5 IU/mg), Lauroyl ethyl arginate, Kolliphor^® RH 40, Centriprep^® centrifugal filter unit and Bovine serum albumin fraction V were purchased from Sigma-Aldrich, Co. (St. Louis, MO, USA). Capmul^® MCM NF and Captex^® 8000 were gifted from Abitec Corporation (Janesville, WI, USA). Human lung cancer cell line (Calu-3) was purchased ATCC^® (Rockville, MD, USA) and Thiazolyl blue tetrazolium bromide, 98% was procured from Acros Organics (Morris Plains, NJ, USA). Transwell inserts were purchased from Corning (Oneonta, NY, USA). Fetal Bovine Serum was purchased from Atlanta Biologicals (Flowery Branch, GA, USA). AimStrip^® Plus glucometer and glucose strips were procured from Germaine Laboratories, Inc (San-Antonio, TX, USA). Human Insulin ELISA Kit was purchased from Crystal Chem (Elk Grove Village, IL, USA). Acetonitrile, methanol, trifluoroacetic acid (TFA) and citrate buffer solution (0.5 M, pH 4.5) were sourced from Fisher Scientific. Dulbecco’s Phosphate-Buffer Saline, pH 7.0 was purchased from Hyclone (Logan, UT, USA). Streptozotocin was procured from Adipogen^® Life Sciences (San Diego, CA, USA). All the purchased components were of analytical grade.

Cells in 2D or 3D aggregates were washed with phosphate-buffered saline (PBS) and then incubated in Krb for 45 min at 37 °C with low glucose (2 mM) to remove residual insulin. Cells were incubated in Krb with low glucose for 90 min and supernatant collected. Then clusters were incubated in Krb with high glucose (20 mM or as indicated in figure) for 90 min and supernatant collected. Human insulin in the collected supernatants was measured using a human Insulin ELISA kit (Crystal Chem, Zaandam, Netherlands).