Ultrasensitive rat insulin elisa kit

The effect of standardized methanolic Ficus deltoidea varieties on insulin secretion was evaluated using clonal pancreatic beta cell line. BRIN BD11 were seeded at 2.5 x 10⁵ cells/ml in 12 well plate and incubated for 24 hours at 37°C. Next, the cells were washed thrice with buffer and preincubated with Krebs-Ringer bicarbonate buffer for 40 minutes and continued for 60 minutes of incubation with the extracts (10-1000 μg/ml) or glybenclamide (10-2000 μg/ml). Ultrasensitive Rat Insulin ELISA kit (Mercodia AB, Sweden) was used to determine insulin concentration [5 (link)].

Plasma insulin was measured using an Ultrasensitive Rat Insulin ELISA kit (Mercodia AB, Uppsala, Sweden, #10-1251-10), plasma GLP-1 using a Rat GLP-1 ELISA kit (EMD Millipore, MA, USA, #EZGLP1T-36 K), plasma leptin using a Rat Leptin ELISA kit (Abcam, Cambridge, UK, #ab100773), plasma alanine transaminase (ALT) using a Rat ALT Simplestep^® ELISA kit (Abcam, Cambridge, UK #ab264579), and plasma aspartate transaminase (AST) using a Rat AST Simplestep^® ELISA kit (Abcam, Cambridge, UK, #ab263883) according to the manufacturer’s instructions.

To study the in vitro effect of ghrelin and/or cytokines on insulin secretion, INS-1E cells or isolated rat islets were cultured in 5.5 mM glucose medium for 24 h, to reduce basal insulin secretion. Subsequently, ghrelin with or without cytokines was added to culture media. Cells were then washed three times with low glucose (2.2 mM) HBSS/Krebs buffer (HBBS), and supernatants collected to determine basal insulin secretion. Afterwards, a high glucose concentration (22 mM) HBBS was added to cultures, and cells were incubated for 90 min. Supernatants were collected again to measure levels of glucose-induced insulin secretion.
Supernatants' insulin levels were determined using a commercially available Ultrasensitive Rat Insulin ELISA kit (Mercodia AB, Uppsala, Sweden) following manufacturer's instructions.

Diabetes was induced in 50 overnight-fasted rats with free access to water by a single IP injection of freshly prepared streptozitocin in ice-cold normal saline at a dose of 55 mg/kg. After 72 h, the rats with a blood glucose level of 15 mM were considered diabetic and used in the experiment. Diabetic rats were divided into seven groups of six rats each (n = 6). Group I consisted of normal rats that received 10 ml/kg of vehicle. Group II consisted of diabetic rats that received 10 ml/kg of vehicle. Group III of diabetic rats served as positive control and received 500 mg/kg metformin. Groups IV, V, VI and VII consisted of diabetic rats treated with 1000 mg/kg of petroleum ether, chloroform, methanol and water extracts, respectively. The treatment was given twice a day for fourteen days. Separate groups of diabetic rats were also treated with 500 mg/kg or 250 mg/kg of the most active extract (methanol extract), administered as described above. Tail vein blood samples were collected on days 1, 3, 6, 9, 12 and 15 for blood glucose analysis, and the body weights were taken [20 ]. The blood samples collected were also used to analyze the fasting plasma insulin levels using the Mercodia Ultra-sensitive Rat Insulin ELISA kit (Sweden). The animals from vehicle and.

INS-1 cells were cultured using the same coculture conditions mentioned previously. After 18 hours of incubation with 5.6 mM glucose medium (RPMI 1640 without glucose (R1383, Sigma) + glucose (Solarbio) + coculture components mentioned above), secretion assays were performed. Adherent cells and pseudoislets were first preincubated for 60 min at 37°C in Krebs-Ringer bicarbonate HEPES buffer (KRBH) (115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl₂, 1.2 mM MgSO₄, 10 mM NaHCO₃, and 20 mM HEPES) containing 1.1 mmol/l glucose supplemented with 1 mg/ml bovine serum albumin (BSA) (Solarbio) [15 (link)]. Following preincubation, they were then exposed to 1.1 mM or 20 mM glucose with or without 10 μM carbachol (Meilun) or 10 μM atropine sulfate (Meilun) for 60 min. The supernatants were then collected from each well and secreted insulin was determined using the Ultrasensitive Rat Insulin ELISA kit (Mercodia, Sweden) according to the manufacturer's protocol. Insulin secretion data were normalized to total insulin content of the cells collected from each well using RIPA buffer (Beyotime). Total insulin content also was determined by ELISA (Mercodia) [16 (link)].

After 2 weeks of treatment, a blood sample was taken between 12 and 2 pm (i.e. at least 18 hours after the previous dosage of metformin), after a 4-hour fast, for determination of post-therapy plasma glucose and insulin concentrations. Plasma glucose concentrations were determined using an automatic glucometer (Freestyle, Abbott, IL, USA). Plasma insulin concentrations were determined with an ultrasensitive rat insulin ELISA kit (Mercodia, Uppsala, Sweden).

To assess the levels of circulating insulin in the blood we used the Ultrasensitive Rat Insulin ELISA kit (Mercodia AB, Uppsala, Sweden). Briefly, blood was collected from the tail vein and centrifuged at 3000 ×g for 10 min to obtain serum which was immediately processed, according to the manufacturer’s protocol.