For insulin secretion, cells were plated in triplicate for each condition. At approx. 100% confluency INS-1 832/13 cells were gently washed with freshly made Secretion Assay Buffer (SAB) pH 7.2, with 2.8 mm glucose (SAB: 114 mm NaCl, 4.7 mm KCl, 1.2 mm KH2PO4, 1.16 mm MgSO4, 20 mm HEPES, 25.5 mm NaHCO3, 2.5 mm CaCl2 and 0.2% BSA). The cells were then pre-incubated for 2 h at 37 °C in fresh SAB supplemented with 2.8 mm glucose and then stimulated for 1 h in 1 mL SAB (containing 2.8 or 16.7 mm glucose as marked in the figures) at 37 °C, 5% CO2. Five hundred microlitre of SAB was collected from each well. Insulin levels were measured according to the manufacturer using Coat-a-Count RIA (Millipore Corporation, Bedford, MA, USA). Insulin secretion measurements were normalized to total protein from each well.
Coat a count ria
Manufactured by Merck Group
Sourced in United States
Coat-a-Count RIA is a radioimmunoassay (RIA) kit developed by Merck Group. It is designed to measure the concentration of specific analytes in biological samples using a radioactive tracer. The kit provides the necessary reagents and protocols to perform the RIA analysis, but its detailed functionality and intended use are not provided in this factual, unbiased description.
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5 protocols using coat a count ria
1
Insulin Secretion Assay in INS-1 832/13 Cells
2
Insulin Signaling Protein Extraction and Quantification
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INS-1 832/13 cells in 24 well plates were washed in ice-cold PBS and incubated on ice for 15 min in RIPA buffer (Radio Immuno Precipitation Assay; 150 nm NaCl, 1% TritonX-100, 0.1% SDS, 50 mm Tris-Cl, pH 8 and EDTA-free protease inhibitor; Roche, Branchburg, NJ, USA). The cells were then loosened by pipetting, transferred to pre-cooled tubes and centrifuged at 4 °C for 15 min at 14 000 g . The supernatant was then transferred to new tubes and stored at −20 °C. Analysis of protein content in cell homogenates was made with BCA assay (Pierce, Rockford, IL, USA) and analysed on the BioRad Model 6870 Microplate Reader (BioRad, Hercules, CA, USA). The insulin content in the samples was determined using Coat-a-Count RIA (Millipore Corporation, Bedford, MA, USA).
3
INS-1 832/13 Cell Lysis and Insulin Quantification
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Prior to protein extraction, the INS‐1 832/13 cells were washed in ice‐cold PBS and incubated on ice for ~15 min in RIPA buffer (Radio Immunoprecipitation Assay); 150 nM NaCl, 1% TritonX‐100, 0.1% SDS, 50 mM Tris‐Cl, pH 8 and EDTA‐free protease inhibitor (Roche, NJ, USA). The resulting cell lysate was harvested, transferred to pre‐cooled tubes, and centrifuged at 4°C for 15 min at 14.000 g. The supernatant was carefully transferred to new tubes and stored at −20°C. Protein content of the supernatant was analyzed using the BCA assay (Pierce®BCA Protein Assay Kit #23227, IL, USA) and BioRad Model 6870 Microplate Reader. The insulin content in the samples was determined using Mercodia High Range Rat Insulin ELISA (# 10‐1145‐01), Coat‐a‐Count RIA (Millipore Corporation, MA, USA), or Mercodia human Insulin ELISA (#10‐1113‐0).
4
Glucose-Stimulated Insulin Secretion Assay
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For glucose‐stimulated insulin secretion assay, cells were plated in triplicate for each condition and the assay was performed as described (Salunkhe et al. 2015 ). For K+‐induced insulin secretion the secretion buffer contained 50 mmol/L KCl (adjusted by reducing equimolar amount of NaCl). Insulin secretion measurements were normalized to total protein/well. Protein extraction was performed using RIPA buffer as previously described (Salunkhe et al. 2015 ). Protein content in cell homogenates was analyzed using BCA assay (Pierce®BCA Protein Assay Kit #23227, IL). Secreted and total insulin were measured using Coat‐a‐Count RIA (Millipore Corporation, MA) and Mercodia insulin ELISA (Mercodia, Uppsala, Sweden).
5
Insulin Secretion Assay in INS-1 and EndoC-βH1 Cells
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For insulin secretion assay, INS‐1 832/13 cells were plated in triplicate for each condition and the assay was performed as previously described.34 Stimulation of insulin secretion was performed with 2.8 mM glucose, 16.7 mM glucose, 16.7 mM glucose + 2.5 μM forskolin, or 2.8 mM glucose + 50 mM K+. In the latter case NaCl2 was substituted with KCl in equimolar ratio. Prior to insulin secretion in EndoC‐βH1 cells, the cells were transferred to a complete glucose starvation medium containing 2.8 mM for 18 h and insulin secretion assay was performed as previously reported.35 Secreted insulin levels were measured using Mercodia High Range Rat Insulin ELISA (# 10‐1145‐01), Mercodia human Insulin ELISA (#10‐1113‐0) or Coat‐a‐Count RIA (Millipore Corporation, MA, USA). Insulin secretion measurements were normalized to total protein content from the same well.
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