Insulin elisa kit

Serum samples from miR-193b AAV-treated C57 and AAV-hsa-sgmiR-193b-treated db/db mice were used to measure blood glucose and insulin levels via OneTouch glucometer and test strips (LifeScan) or an insulin ELISA kit (catalogue no.: ab277390; Abcam, Cambridge, MA, USA), respectively (see ESM Methods). For refed glucose samples, miR-193b AAV-treated C57 and sgmiR-193b AAV-treated db/db mice (and their controls) were fasted overnight and then food was replaced for 2 h before determination of refed blood glucose (see ESM Methods).

INS-1 cells were seeded in a 96-well plate with further culturing for 24 h, and the cells were treated with 3.3 mM glucose (basal glucose) or 16.7 mM glucose (stimulatory glucose) for 1 h. After that, the insulin level was measured by Insulin ELISA kit (Abcam, UK) according to the manufacturer's instructions.

RIN-5F cells were seeded in 24-well plates at 2 × 10⁵ cells/well and incubated at 37 °C and 5 % CO2. After incubation for 24 h, the medium was removed from the wells, and the cells were washed twice with fresh medium containing low glucose (6.25 mM) or high glucose (12.5 mM). Afterward, the cells were incubated at 37 °C for 3 h with low glucose or high glucose medium, supplemented with 1 % FBS and treated with low or high concentrations of UD (1.5 and 3 mg/mL). Then, the aliquots in all wells were collected to determine the concentration of insulin in the media with the use of ELISA kit (Insulin ELISA kit, Ab100578, Abcam, Cambridge, UK) according to the manufacturer’s instructions. The insulin secretion levels at different concentrations of saffron were assessed by comparing them with the control insulin secretion level. The 0 concentration of extract (untreated cell) was considered as the control. The experiment was conducted in triplicate, and the data were presented as mean ± SD.

Fasting serum insulin concentrations were measured in 20-week old TRIM31^−/− and WT mice by using an insulin ELISA kit (Abcam, Cambridge, UK). Insulin resistance was evaluated by the HOMA-IR score, calculated by fasting serum insulin (μU/ml)×fasting plasma glucose (mmol/l)/22.5.