Dulbecco s modified eagle s medium dmem ham s f12

Mouse islets were isolated from db/m and db/db mice (12 weeks old and 6 mice each group, the blood glucose levels were greater than 13mmol/l) by type IV collagenase (1 mg/ml; Sigma, CA, USA) digestion of the exocrine pancreas followed by purification on Histopaque (Sigma, CA, USA) density gradients. The isolated islets were maintained in culture for up to 48 hours at 37°C (95% air/5% CO₂) [24 (link)], with the majority of islets attaching to the dish within 3 to 7 days. Cultures were re-fed when the majority of islets had attached and thereafter as needed to replenish nutrients and remove debris. “Passage 0” is defined as 10 to 14 days after the islets were placed in culture at a time when the cultures were nearly confluent with stellate cells. Beginning at passage 0, cells were harvested with trypsin and sub-cultured (1:2) every 3-4 days. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 (1: 1 v/v) (Sigma, CA, USA) containing 10% (v/v) fetal calf serum (FCS) and used from passage 3-8.

Mouse islets were isolated from mice (8 weeks old) by type IV collagenase (1 mg/ml; Sigma, CA, USA) digestion of the exocrine pancreas followed by purification on Histopaque 1077 (Sigma, CA, USA) density gradients. The isolated islets were cultured in RPMI-1640 medium containing 10% (v/v) foetal bovine serum (FBS) and penicillin-streptomycin for up to 48 h at 37°C, and the majority of islets attached to the dish within 7 days. “Passage 0” was defined as the time when the cultures were nearly confluent with ISCs. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 (1 : 1 v/v) (Sigma, CA, USA) containing 10% (v/v) FBS and used at passages 3–8. Min6 cells were divided into groups and cultured alone, with ISC supernatant or with exogenous Wnt5a (0.05 μg/ml) (Wnt5a; R&D Systems, UK) for 48 h unless otherwise specified.