Mouse high range insulin elisa kit

The glucose‐sensitive mouse β‐cell line SJ β‐cells were cultured and insulin‐secretion assay was performed as reported earlier (Jia et al., 2015). In brief, insulin secreted into medium and from whole cell lysates was quantified with Mouse High Range Insulin ELISA kit, according to the manufacturer's protocol (Alpco, USA). Cells were incubated for 30 min with low (3.3 mmol/L) or high (16.7 mmol/L) glucose concentration. We normalized secreted insulin by the total cellular protein. All the values were expressed in relation to cells transfected with wild‐type human HDAC4.

MIN6 cells and dissociated islet cells were transfected with miRVec-miR-7, pCDNA3-ciRS-7 and their control plasmids respectively using Lipofectamine 2000 (Invitrogen, Grand Island, NY) for further glucose stimulated insulin secretion (GSIS) assay. The transfection efficiency of the MIN6 and dissociated islet cells were about 30% and 25%, respectively, as evaluated by cotransfected EGFP-expressing plasmid. After 48 h incubation, these cells were washed twice in buffer A (5 mM KCl, 120 mM NaCl, 24 mM NaHCO3, 1 Mm MgCl2, 2 mM CaCl2, 1 mg/ml Ovalbumin, 15 mM HEPES at pH 7.4) and then transferred to buffer A with 3.3 mM glucose for 2 hours to stabilize basal insulin secretion. After MIN6 and Islet cells were rinsed twice with buffer A without glucose, glucose stimulation was performed in buffer A with either 3.3 mM glucose or 16.7 mM glucose for 1 hour. Insulin levels in culture medium and in cell lyses were measured with Mouse High Range Insulin ELISA kit (ALPCO Diagnostics, Salem, NH). Fold changes were calculated between the basal insulin and the stimulated insulin level.