INS-1 cells were plated onto 24-well plates and were grown to confluency prior to assay. The standard tissue culture medium was switched to medium containing 5 mM glucose for 18 h. Insulin secretion was performed in HEPES balanced salt solution (HBSS) (114 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.16 mM MgSO4, 20 mM HEPES, 2.5 mM CaCl2, 25.5 mM NaHCO3 and 0.2% bovine serum albumin, pH 7.2). Cells were washed with HBSS containing 3 mM glucose, followed by a 2 h incubation in the same buffer. Insulin secretion was then measured in static incubations of HBSS containing 3 mM or 15 mM glucose containing 200 μM tolbutamide or diazoxide for 2 h. Insulin levels were measured using the porcine insulin radioimmunoassay (RIA) (Millipore, PI-12K). Human insulin was used for standard curves.
Pi 12k
Manufactured by Merck Group
Sourced in United States
The PI-12K is a laboratory centrifuge that can be used to separate and isolate different components of a liquid sample through centrifugal force. It has a maximum speed of 12,000 rpm and can accommodate a variety of sample sizes and types.
Automatically generated - may contain errors
Lab products found in correlation
Xl 85k, by Merck Group (1 mentions)
Ocpe07 t3, by PerkinElmer (1 mentions)
Ocpg07 t4, by PerkinElmer (1 mentions)
Gm9 glucose analyzer, by Analox Instruments (1 mentions)
Konelab i 20xti, by Thermo Fisher Scientific (1 mentions)
Accu chek aviva, by Roche (1 mentions)
Model 2300, by Yellow Springs Instruments (1 mentions)
6 protocols using pi 12k
1
Insulin Secretion Assay in INS-1 Cells
2
Serum Metabolites and Hormones in Dairy Cattle
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Blood samples were collected in the last 14 days of each Latin square periods from the jugular vein 0.5 h before and 0.5, 1.5, 2.5, 3.5, 5, 7, 9, 11 h after PG administration as OD and morning feeding (CON, TMR, TD) into a tube with polystyrene separating granules covered with a clot activator. After 1 h the samples were rotated in a centrifuge, the serum was separated and frozen at -20 °C for later analysis. Serum was thawed and analysed for NEFA concentration according to Duncomb's colorimetric method (Duncombe 1964) . The concentrations of glucose, triglycerides, β-hydroxybutyric acid (BHBA) and blood urea nitrogen (BUN) were assayed using a Pointe Scientific (Canton, USA) reagent kit (G7518-400, T7531-400, H7587-58, B7550-400). Serum hormone concentrations were analysed using radioimmunoassay (RIA) methods: insulin (PI-12K, Millipore Corporation, Burlington, USA), insulin-like growth factor I (IGF-I; DSL-2800, Diagnostic Systems Lab., Webster, USA), leptin (XL-85K, Millipore Corporation, Burlington, USA), ghrelin (GHRT-89HK, Linco Research, St. Charles, USA), thyroxine, and triiodothyronine (OCPG07-T4, OCPE07-T3, CISBIO International, Codolet, France). Individual voluntary dry matter intake was measured and recorded daily before the morning and afternoon feeding.
3
Plasma Glucose and Insulin Assay Protocol
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Plasma glucose concentrations were determined enzymatically using an automated analyzer (YSI 2700 Select Analyzer, YSI Inc., Yellow Spring, OH). Plasma insulin levels were assayed using a commercially available radioimmunoassay kit (PI-12K, Millipore Sigma, Burlington, MA) validated for horses as previously described (23 (link)). All plasma samples were run in duplicate. Average intra and inter-assay variation for glucose was 0.3 and 3.4%, respectively. Average intra and inter-assay variation for insulin was 7.6 and 7.4%, respectively.
4
Liver Metabolic Profiling in Humans
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Arterial, hepatic portal vein, and hepatic vein whole blood samples collected during the study were analyzed using a variety of standard methods to obtain data regarding hormone and substrate balance across the liver. The arterial and portal blood samples were collected at the same time, followed by the hepatic venous blood sample. This allowed for the transit time of blood across the liver. Plasma glucose samples were immediately measured upon collection using a GM9 glucose analyzer (Analox Instruments Ltd., Stourbridge, UK). Blood lactate, glycerol, alanine, and non-esterified fatty acids were analyzed using enzymatic spectrophotometric methods (see supplement ). Plasma insulin (#PI-12K, MilliporeSigma, Burlington, MA), glucagon (#GL-32K, MilliporeSigma), and cortisol (VUMC Analytical Services in-house primary antibody with I125 cortisol from MP Biomedicals, Santa Ana, CA) were measured by radioimmunoassay. All samples were kept in an ice bath during the experiment and subsequently stored at −80°C until assays were performed. For the determination of [3-3H]-glucose, plasma samples were deproteinized and quantified using liquid scintillation counting (40 (link)). Hepatic glycogen was quantitatively assessed using the amyloglucosidase method (see supplement ).
5
Glucose and Insulin Measurement in Equine CGIT
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Whole blood glucose concentrations during the CGIT procedure were measured immediately after collection using a handheld glucometer (Accu-Chek Aviva, Roche Diagnostic, Indianapolis, IN). Plasma glucose concentrations from samples collected during the feed challenge test were determined with an enzymatic assay (Konelab i 20XTi, Thermo Electron Corp., Waltham, MA). Plasma insulin concentrations were determined with a commercially available radioimmunoassay kit (PI-12K, Millipore Sigma, Burlington, MA) with has previously been validated in horses (21 (link)). All plasma samples were run in duplicate within a single assay. Average intra-assay variation was 1.3 and 5.1% for glucose and insulin, respectively.
6
Plasma Insulin and Glucose Measurement
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