High range rat insulin elisa

To estimate the insulin sensitivity, we measured fasting (12-h fast) plasma glucose and insulin concentration for HOMA-IR calculation. Blood samples were taken from a tail vein and glucose concentration was measured in duplicate using glucometer Accu-Chek (Roche, Germany). To determine insulin concentration, 50 μL of blood sample was taken into heparinized Microvette capillary tube (Sarstedt, Numbrecht, Germany), centrifuged, and measured in duplicate using a solid phase two-site enzyme immunoassay (High Range Rat Insulin ELISA, Mercodia AB, Sweden). The equation for calculating HOMA-IR was as follows: HOMA-IR = (fasting plasma glucose × fasting plasma insulin)/2430, where fasting plasma glucose was in mg/dL and fasting plasma insulin in μU/mL [37 (link)].

Cells transfected with mature miRNAs or siRNA as described above were lysed in 200 μL RIPA buffer after 72 h post-transfection. Total insulin and proinsulin were determined using Mercodia High Range Rat Insulin ELISA and Rat/Mouse Proinsulin ELISA (Mercodia AB, Sweden) respectively according to the manufacturer’s protocol.

INS-1 832/13 cells (150,000 cells per well) were cultured in 24-well plates (Sarstedt, USA) and transfected with siRNA target to Piezo1 for 96 h. The cells were washed with 1 ml secretion assay buffer (SAB) (114 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.16 mM MgSO₄, 25.5 mM NaHCO₃, 2.6 mM CaCl₂, 20 mM HEPES (pH 7.3), 0.2% BSA) with 2.8 mM glucose twice, and pre-incubated in 2.8 mM glucose SAB for 2 h at 37 °C with 5% CO₂ in the incubator. Insulin secretion was measured in static incubations of cells (500 μl SAB containing 2.8 or 16.7 mM glucose for 1 h). In the experiments involving hypotonicity, the concentration of NaCl was reduced to 54 mM NaCl. The supernatant was collected pending later measurements of insulin release. Insulin content was determined after lysing the cells with 200 μl RIPA buffer (see above) followed by shaking on ice for at least 20 min. Insulin concentrations were measured with a high-range rat insulin ELISA (#10-1145-01, Mercodia AB) and normalized to protein content. Protein content was determined using the Pierce™ BCA protein assay kit (#23225, Thermo Fisher Scientific).