Whole SC-β cell clusters or cells attached to culture plates were washed twice thoroughly with PBS. Half of the clusters or an equivalent well of plated cells were immersed in TrypLE for cell counts on the Vi-Cell XR. For the other half of the samples, a solution of 1.5% HCl and 70% ethanol was added to either the clusters in eppendorf tubes or directly onto plated cells. After 15 minutes, the plated cells were pipetted vigorously and transferred to eppendorf tubes. The eppendorf tubes from both clusters and plated cells were kept at −20°C for 72 hours, vortexing vigorously every 24 hours. Samples were then centrifuged at 2100 RCF for 15 minutes. The supernatant of each sample was collected, neutralized with an equal volume of 1 M TRIS (pH 7.5), and quantified using a proinsulin ELISA kit (Mercodia, 10–1118-01) and a human insulin ELISA kit. Proinsulin and insulin secretion were normalized to the viable cell counts.
Proinsulin elisa kit
Manufactured by Mercodia
Sourced in Sweden, Japan
The Proinsulin ELISA kit is a laboratory assay developed by Mercodia to quantify proinsulin levels in biological samples. The kit utilizes the enzyme-linked immunosorbent assay (ELISA) technique to detect and measure proinsulin concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Human insulin elisa kit, by Mercodia (2 mentions)
Cyquant direct cell proliferation assay, by Thermo Fisher Scientific (1 mentions)
Insulin human alphalisa detection kit, by PerkinElmer (1 mentions)
Enspire alpha plate reader, by PerkinElmer (1 mentions)
Rat mouse insulin elisa kit, by Crystal Chem (1 mentions)
Liraglutide, by Novo Nordisk (1 mentions)
5 protocols using proinsulin elisa kit
1
Quantifying Proinsulin and Insulin Secretion
2
Quantifying Proinsulin and Insulin Secretion
3
Quantifying Glucose-Stimulated Insulin Secretion
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Silenced EndoC-βH1 cells were starved overnight at 48 h post-transfection in culture medium containing 2.8 M glucose followed by 30 min incubation in 0 mM glucose. Insulin secretion was initiated through static incubations in the indicated glucose or secretagogues conditions for 1 h. Insulin containing supernatant was collected, and cells were lysed in ice-cold acid ethanol to release intracellular insulin. Insulin was quantified using the Insulin (human) AlphaLISA Detection kit and the EnSpire Alpha Plate Reader (both Perkin Elmer) based on 1:10 and 1:200 dilutions for supernatant and insulin content, respectively. Intracellular proinsulin was quantified using the Proinsulin ELISA kit (Mercodia). Secreted insulin was normalized to the level of intracellular insulin content or cell count, which was measured before cell lysis using the CyQUANT direct cell proliferation assay (Invitrogen).
4
Insulin and Proinsulin Quantification
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Total insulin and proinsulin content were measured using rat/mouse insulin ELISA kit (Crystal Chem, IL) and proinsulin ELISA kit (Mercodia, Uppsala, Sweden) respectively from batches of ten size-matched islets after acid-ethanol extraction [12 (link)].
5
Glucose-Stimulated Insulin Secretion in Mice
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Blood glucose levels and serum proinsulin or insulin levels in mice were determined using Glutest Neo Super (Sanwa Kagaku Kenkyusho, Nagoya, Japan) and proinsulin ELISA kit (10-1232-01; Mercodia) or insulin ELISA kit (M1102; Morinaga, Yokohama, Japan), respectively. For evaluating GSIS from the islets of the C57BL/6J mice after treatment with or without 1 mmol/L imeglimin and 100 nmol/L liraglutide (Novo Nordisk), 10 isolated islets were incubated in Krebs-Ringer bicarbonate buffer (pH 7.4) with 3.9 mmol/L, 11.1 mmol/L, or 16.7 mmol/L glucose for 60 min (preincubation with 3.9 mmol/L glucose with or without imeglimin for 60 min). The islets were extracted with acid ethanol, and the insulin/proinsulin concentration in the assay buffer and the insulin/proinsulin content in the islets were measured using an insulin ELISA kit.
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