Ultrasensitive c peptide elisa kit

Insulin and C-peptide secretion was measured in the culture medium of DIPCs and DIPC spheroids by static incubation for 2 days in differentiation culture medium containing 5.5 mM glucose. Additionally, DIPCs and spheroids were washed and lysed using RIPA lysis buffer to detect intracellular insulin contents [24 (link)]. Prior to glucose stimulation of cells, residual insulin released from cells was removed by incubating in serum- and glucose-free Krebs buffer. The tubes were incubated at 37 °C for 1 h with shaking; then, the medium was completely removed by centrifugation and replaced with Krebs buffer supplemented with 3 mM glucose for 1 h. After collecting the medium, the cells were incubated with Krebs buffer containing 30 mM glucose for 1 h. Supernatants were collected. The stimulation index was calculated by dividing the insulin concentration of the supernatant in response to the 30 mM glucose incubation by that of the supernatant from the 3 mM glucose incubation [25 (link)]. Insulin and C-peptide levels were measured using a commercial ultrasensitive insulin ELISA kit (ALPCO, Salem, NH, USA) and ultrasensitive C-peptide ELISA kit (Mercodia, Uppsala, Sweden), respectively.

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.

To measure the insulin and C-peptide contents in IPCs, cells were washed and lysed with RIPA lysis buffer [21 (link)]. Insulin and C-peptide levels were measured using a commercial ultrasensitive insulin ELISA kit (80-INSHUU-E01.1 ALPCO, Salem, NH, USA) and ultrasensitive C-peptide ELISA kit (10-1141-01, Mercodia, Uppsala, Sweden), respectively. Also, insulin secretion was measured in the culture medium of IPCs and IPC sheets by static incubation for 2 days in differentiation culture medium containing 5.5 mM glucose. Insulin and C-peptide contents were ascertained in cells from four different donors. Prior to glucose stimulation of the cells, any residual insulin released from the islets was removed by incubation in serum- and glucose-free RPMI 1640 medium. The tubes were kept at 37 °C for 1 h with shaking, and then, the medium was completely removed by centrifugation and replaced with serum-free RPMI 1640 medium supplemented with 2.8 mM glucose for 1 h. Following collection of the medium, the cells were then incubated with serum-free RPMI 1640 containing 28 mM glucose for an additional 1 h. The supernatant from each sample was collected. The assays were performed in triplicate. The stimulation index was calculated as a ratio between the insulin secreted at high and low glucose media [22 (link)].