For all tests, mice were individually housed. For the glucose tolerance test (GTT), mice were fasted for 5 h in the morning before they were given an intraperitoneal injection of glucose (2 g/kg body weight) dissolved in a 0.9% saline solution. For the insulin tolerance test (ITT), overnight fed mice were fasted for 2 h in the morning before they were given an intraperitoneal injection of insulin (1 U/kg body weight, Actrapid, Novo Nordisk, Bagsværd, Denmark). Blood was collected from the tail vein at 0, 20, 40, 60, 90 and 120 min in the GTT and 0, 20, 40 and 60 min in the ITT. Blood glucose concentrations were determined by using a glucometer (Contour XT, Bayer, Germany). For the GTT, plasma insulin concentrations were determined at 0, 20, and 40 min by using an enzyme-linked immunosorbent ELISA assay (Cat. No. 80-INSMSU-E10, ALPCO) according to the manufacturer's instructions.
80 insmsu e10
Manufactured by ALPCO
Sourced in United States
The 80-INSMSU-E10 is a laboratory instrument designed for insulin measurement. It utilizes an electrochemical detection method to quantify insulin levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Contour xt, by Bayer (1 mentions)
Actrapid, by Novo Nordisk (1 mentions)
Micro hematocrit heparinized capillary tubes, by Thermo Fisher Scientific (1 mentions)
Human insulin elisa kit, by Merck Group (1 mentions)
U 13c6 glucose tracer, by Cambridge Isotopes (1 mentions)
Dgcg0, by R&D Systems (1 mentions)
Infinity triglyceride reagent, by Thermo Fisher Scientific (1 mentions)
11 protocols using 80 insmsu e10
1
Glucose and Insulin Tolerance Tests in Mice
2
Quantifying Insulin and Proinsulin in Transgenic Mice
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Milk samples from the transgenic mice and non-transgenic mice were collected during early (3–5 d), middle (9–11 d), and late (15–17 d) lactation using a Medela Freestyle pump (McHenry, IL). Just prior to milking, 5 I.U. of oxytocin was injected intraperitoneally to the mice. The milk samples were defatted by centrifugation at 4°C for 15 min at 10,000 g. The resulting skim milk was diluted one million fold, and the human proinsulin concentrations were then determined with a Human Insulin ELISA Kit (RAB0327) from Sigma according to the manufacturer's instructions. Tail blood samples (40 μl) were drained into heparinized microhematocrit capillary tubes (Fisher Scientific, Pittsburgh, PA), transferred to centrifuge tubes, and centrifuged at 4°C and 2,000 g for 10 min. The resulting supernatant (plasma) was saved and used for ELISA analysis. Two different ELISA kits were used: 1) a Human Insulin ELISA Kit (RAB0327, Sigma), which is specifically used to measure human insulin and proinsulin; and 2) a mouse Ultrasensitive Insulin ELISA kit (80-INSMSU-E10, Alpco, Salem, NH), which has 147% and 0.27% cross-reactivity against human insulin and proinsulin, respectively.
3
Glucose-Stimulated Insulin Secretion Assay
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As an exploratory analysis, one week after GTT and ITT, animals underwent intraperitoneal glucose-stimulated insulin secretion (GSIS) testing (PND 160–170: eTRF males = 4, eTRF females = 4, AL males = 5, and AL females = 8). At ZT2, animals were placed in a clean cage without food and with unrestricted access to water. After a 6-hour fast, animals were lightly anesthetized with isoflurane via drop jar and a baseline blood sample was collected via retro-orbital bleed with a heparinized capillary. Following baseline blood collection, an intraperitoneal injection of D-glucose (1.0 g/kg lean mass) was given. After 15 minutes, animals were lightly anesthetized in the same manner and another blood sample was collected. Blood samples were allowed to clot on wet ice (∼20 minutes) and then were spun down in a cold centrifuge (4°C, Eppendorf microcentrifuge, model 5415R) for 20 minutes at 2000g. Serum was pipetted off and stored at −80°C until analysis. Serum insulin was assessed via a commercially available ELISA kit (ALPCO 80-INSMSU-E10). Serum insulin was assessed in 5 μL samples and read via a colorimetric assay.
4
Glucose Metabolism Kinetics in Mice
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To measure glucose metabolism, mice were orally administered with [U-13C6]glucose tracer (1.5 g/kg, Cambridge Isotope Laboratories, Andover, MA, USA) following 6 h fasting. Blood was collected from the tail tip at a time prior to −120 min, and 10, 20, 30, and 60 min after the oral glucose tracer administration. Plasma glucose concentration was immediately measured using a glucometer (GM9, Analox Instrument, Stoubridge, UK). Plasma insulin concentration was measured using an insulin ELISA kit, in accordance with manufacturer’s instructions (80-INSMSU-E10, ALPCO, Salem, MA, USA). Total rate of glucose appearance (Ra) was calculated based on the single pool kinetic using an integral approach [29 ].
where 60 is the end time point of MPE during glucose tolerance, 0 is the baseline value over MPE, and dose is the amount of glucose in the oral gavage. Insulin resistance was calculated using homeostasis model assessment of insulin resistance (HOMA-IR), and Matsuda insulin sensitivity index was used for an index of insulin sensitivity [47 (link)].
where 60 is the end time point of MPE during glucose tolerance, 0 is the baseline value over MPE, and dose is the amount of glucose in the oral gavage. Insulin resistance was calculated using homeostasis model assessment of insulin resistance (HOMA-IR), and Matsuda insulin sensitivity index was used for an index of insulin sensitivity [47 (link)].
5
Oral Glucose Tolerance Test in Mice
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The mice were fasted for 6 h. At the start of the oral glucose tolerance test, the body weight was measured, and a baseline plasma was collected via the tail vein for the determination of fasting glucose and insulin prior to an oral gavage of a 1.5 g/kg body weight glucose (1:1 ratio of [U-13C6]glucose and unlabeled glucose). Plasma was collected at 0, 10, 20, and 60 min post-gavage using capillary tubes. Plasma glucose concentration was measured immediately using a glucometer (GM9, Analox Instruments, Stourbridge, UK). Plasma insulin was determined using an insulin ELISA kit (80-INSMSU-E10, ALPCO, Salem, MA, USA). The homeostasis model assessment IR (HOMA-IR) index and Matsuda insulin sensitivity index were calculated as described in the previous study [64 (link)]. The area under the curve (AUC) was calculated for each adjacent time point of plasma glucose concentration, enrichment, hepatic glucose output, and insulin concentration.
6
Plasma Insulin Measurement and HOMA-IR
7
Cytokine and Inflammatory Biomarker Profiling
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The levels of interleukin (IL)-10 (M1000B), IL-1β (MHSLB00), and IL-6 (M6000B), monocyte chemotactic protein (MCP-1; MJE00B), tumor necrosis factor (TNF)-a (MTA00B), and C-reactive protein (CRP; DY1829) in plasma were monitored using ELISA kits (R&D Systems, USA). The concentrations of glucagon (DGCG0; R&D Systems, USA) and insulin (80-INSMSU-E10; ALPCO, USA) were determined by ELISA kits according to the manufacturer’s instructions.
8
Plasma Insulin Measurement and HOMA-IR
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Plasma was collected during euthanasia, as described above, and insulin was measured by ELISA (Alpco; 80-INSMSU-E10) according to manufacturer’s instructions (Stanley et al., 2016 (link)). Homeostasis model assessment of insulin resistance scores were then calculated (HOMA-IR = plasma glucose [mmol/L] × plasma insulin [U/mL]/22.5).
9
Metabolic Evaluation in Fasted Mice
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Mice were fasted for four hours beginning at 6 AM on the day of the experiment. Blood samples obtained from the tail vein were used for insulin and triglyceride measurements. Following administration of glucose (2 g per kg oral gavage), glucose levels were measured immediately before and 15, 30, 60 minutes after injection. Plasma insulin content was determined by using Insulin (mouse) ultra-sensitive EIA kit (80-INSMSU-E10, ALPCO Diagnostics).
At termination, mice were euthanized, the blood was collected by cardiac puncture, and various tissues were harvested for histological analysis. Plasma triglyceride levels were measured by using Infinity Triglyceride Reagent (TR22321, Thermo Scientific). Serum leptin levels were measured using Mouse/Rat Leptin ELISA kit, Ref# RD291001200R.
At termination, mice were euthanized, the blood was collected by cardiac puncture, and various tissues were harvested for histological analysis. Plasma triglyceride levels were measured by using Infinity Triglyceride Reagent (TR22321, Thermo Scientific). Serum leptin levels were measured using Mouse/Rat Leptin ELISA kit, Ref# RD291001200R.
10
Plasma Glucose and Insulin Analysis
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