Htrf insulin assay kit

Primary murine islets were cultured overnight post-isolation before the perfusion assays. For each assay, perfusion chambers were loaded with 150 islet equivalents [IEQs] (approximately 100 size-matched islets) islets. Islets were first perfused with KRB buffer containing 2.8 mM glucose for 30 min, followed by perfusion with KRB buffers containing 2.8 mM glucose, 16.8 mM glucose and 2.8 mM glucose for 25, 30 and 20 min, respectively, under a 1 ml/min flow rate. Fractions were collected every minute. Islets were collected from the perfusion chamber after the assay to obtain the total protein content via the BCA protein assay. Insulin secretion at each time point was quantified using an HTRF insulin assay kit (Cisbio, 62INSPEC) according to the manufacturer’s instructions, and normalized to the total protein content.

Differentiated ES-DBCs, EN cells at stage 5, or human Islets were subjected to a GSIS assay. The differentiated cells and human islets (~ 50 islets) were washed 2 times with KRB (Krebs-ringer bicarbonate pH 7.2; (112 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 5 mM NaHCO3, 20 mM HEPES, and 0.1% BSA)) and then incubated with low glucose KRB for 60 minutes at 37°C. Next, the cells were incubated with KRB containing low glucose (2.8 mM), high glucose (16.5 mM), and both high glucose and KCl (16.5 mM glucose⁺30 mM KCL) for 30 minutes sequentially. To measure insulin and C-peptide content in the ES-DBCs at stage 5, the cells were suspended in Tris-EDTA (pH 7.4) on ice and then briefly sonicated until the cell membranes disappeared. The cell debris was removed via centrifugation and the intracellular insulin content was measured from the supernatant. Human insulin levels were measured using a Homogenous Time Resolved Fluorescence (HTRF) insulin assay kit (Cisbio), according to manufacturer’s instruction. C-peptide was measured using Ultra-Sensitive C-peptide ELISA kit (Mercodia), according to manufacturer instructions. All the Insulin and C-peptide measurements were performed on the PHERAstar FS (BMG Labtech). For normalization, the total protein contents of the cell lysates were measured using a Bradford assay.

Glucose stimulated insulin secretion (GSIS) was determined as previously described in detail [46 (link)]. Islets were cultured for 2 days in complete RPMI 1640 medium with 10% FBS in the absence or presence of various stimulants. Then 10 islets/well were selected and transferred to 48-well plates. The islets were then incubated in Krebs–Ringer bicarbonate HEPES buffer (KRBH, 135 mM NaCl, 3.6 mM KCl, 0.5 mM NaH₂PO₄, 1.5 mM CaCl₂, 2 mM NaHCO₃, 10 mM HEPES and 0.1 % BSA, pH 7.4) and 0.1% fatty acid-free BSA for 1 h and then incubated in KRBH buffer containing 2.8 mM or 16.7 mM glucose for another hour at 37 °C. One hundred microliters of supernatant was removed for determination of insulin levels. The insulin content was detected via a HTRF insulin assay kit (Cisbio, 62INSPEC). Cell membrane integrity, which is indicated by LDH, was measured using a CytoTox-ONE™ Homogeneous Membrane Integrity Assay kit (Promega, Madison, WI; #G7891) according to the manufacturer’s instructions.