Glucose and insulin tolerance tests were performed on fasted mice expressing DREADDs (hM3DGq or hM4DGi) to assess the role of refeeding-responsive DMH neurons. To activate DREADDs, clozapine (CLZ) solution (0.1 mg/kg; Cat# C6305, Sigma–Aldrich, St. Louis, MO, USA) was injected (i.p.) 10 min or 30 min before glucose or insulin tolerance tests. We chose CLZ instead of clozapine N-oxide (CNO), because CLZ can activate DREADD receptors at a lower concentration than CNO [20 (link)]. For the glucose tolerance test, the animals were fasted overnight and injected with glucose (2 g/kg; i.p.). Blood glucose levels were measured with a handheld glucose meter (Nipro Free Style, Nipro, Osaka, Japan) before injection of CLZ (−10 or −30 min) and glucose (0 min), as well as 15, 30, 60, and 120 min after glucose injection. The insulin tolerance test was performed on ad libitum-fed mice. The mice were injected with 0.75 U/kg insulin (Novo Nordisk, Bagsværd, Denmark). Blood glucose was measured before injection of CLZ (−10 or −30 min) and insulin (0 min) as well as at 15, 30, 60, and 120 min after insulin injection.
Free style
Manufactured by Nipro
Sourced in Japan
The FreeStyle is a compact and portable lab equipment designed for accurate and reliable testing. It features a user-friendly interface and advanced technology to ensure consistent performance. The core function of the FreeStyle is to facilitate efficient and precise measurements for a variety of laboratory applications.
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Lab products found in correlation
Insulin, by Novo Nordisk (3 mentions)
Lactate pro, by Arkray (3 mentions)
Chemiluminescent enzyme immune assays, by Fujirebio (3 mentions)
Lactate pro 2, by Arkray (3 mentions)
Enzyme linked immunosorbent assay, by Cayman Chemical (2 mentions)
Human il 6 quantikine hs, by R&D Systems (1 mentions)
Humulin r, by Eli Lilly (1 mentions)
Hs600b, by R&D Systems (1 mentions)
Lactate pro2 lt 1730, by Arkray (1 mentions)
Ek 067 52, by Phoenix Pharmaceuticals (1 mentions)
Enzyme linked immunosorbent assay kit, by R&D Systems (1 mentions)
Human il 6 quantikine hs elisa kit, by R&D Systems (1 mentions)
Opti cca ts, by Sysmex (1 mentions)
20 protocols using free style
1
Neuromodulation Regulates Glucose Homeostasis
2
DREADD-Mediated Glucose and Insulin Regulation
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Glucose and insulin tolerance tests were performed on fasted mice expressing DREADDs (hM3DGq or hM4DGi) to assess the role of refeeding-responsive DMH neurons. To activate DREADDs, clozapine (CLZ) solution (1 mg/kg; Toronto Research Chemicals, North York, ON, Canada) was injected (i.p.) 30 min before glucose or insulin tolerance tests. For the glucose tolerance test, animals were fasted for 16 h and injected with glucose (2 g/kg; i.p.). Blood glucose levels were measured with a handheld glucose meter (Nipro Free Style, Nipro, Osaka, Japan) before injection of CLZ (-30 min) and glucose (0 min), as well as 15, 30, 60 and 120 min after glucose injection. The insulin tolerance test was performed on ad libitum-fed mice. The mice were injected with 0.75 U/kg insulin (Novo Nordisk, Bagsvaerd, Denmark). Blood glucose was measured before injection of CLZ (-30 min) and insulin (0 min), as well as 15, 30, 60 and 120 min after insulin injection.
3
Hypothalamic Regulation of Glucose Homeostasis
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A glucose tolerance test was performed on ad libitum fed or fasted mice. The ad libitum fed mice were used for assessing the effects of inhibitors. The fasted mice were used for assessing the phenotype of mice with knockdown of pla2g4a in Sf1-neurons (cPLA2KD Sf1 ). To assess the effects of inhibitors, methyl arachidonyl fluorophosphonate (MAFP; 20 μM, 300 nL in each side), indomethacin (140 μM, 300 nL in each side), or vehicle were injected into both sides of the hypothalamus through a double-cannula.
Glucose solution was then injected i.p. (2 g/kg) 30 min after intrahypothalamic injection.
To assess the phenotype of cPLA2KD Sf1 mice, animals were fasted for 16 hours and injected with glucose (2 g/kg) i.p.. Blood glucose levels were measured by a handheld glucose meter (Nipro Free style, Nipro, Osaka, Japan) before injecting inhibitors (-30 min) or glucose (0 min), and measured at 15, 30, 60 and 120 min after glucose injection.
An insulin tolerance test was performed in ad libitum fed mice. The mice were i.p. injected with 0.5 U/kg insulin (Novo Nordisk, Bagsvaerd, Denmark). Blood glucose was measured before injecting inhibitors (-30 min) and glucose (0 min), and measured 15, 30, 60 and 120 min after glucose injection.
Glucose solution was then injected i.p. (2 g/kg) 30 min after intrahypothalamic injection.
To assess the phenotype of cPLA2KD Sf1 mice, animals were fasted for 16 hours and injected with glucose (2 g/kg) i.p.. Blood glucose levels were measured by a handheld glucose meter (Nipro Free style, Nipro, Osaka, Japan) before injecting inhibitors (-30 min) or glucose (0 min), and measured at 15, 30, 60 and 120 min after glucose injection.
An insulin tolerance test was performed in ad libitum fed mice. The mice were i.p. injected with 0.5 U/kg insulin (Novo Nordisk, Bagsvaerd, Denmark). Blood glucose was measured before injecting inhibitors (-30 min) and glucose (0 min), and measured 15, 30, 60 and 120 min after glucose injection.
4
Exercise-Induced Changes in Iron Metabolism
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Blood samples were collected from an antecubital vein before exercise (before the start of the exercise period), immediately after exercise, and 3 h after exercise. The samples were centrifuged (3,000 rpm, 4°C), and serum and plasma samples were stored at -80°C until subsequent analyses. Serum ferritin, iron, and hepcidin levels were measured. Ferritin and iron levels were measured at a clinical laboratory (SRL Inc., Tokyo, Japan), and hepcidin levels were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA). Plasma IL-6 levels were measured using an ELISA kit (Human IL-6 Quantikine HS; R&D Systems). Glucose and lactate levels were measured immediately after the collection of blood using a glucose analyzer (Freestyle, Nipro Co., Osaka, Japan) and a lactate analyzer (Lactate Pro; Arkray Co., Kyoto, Japan), respectively.
5
Metabolic and Inflammatory Responses Assessment
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Initial blood sample was taken from the antecubital vein after 30 min of rest. Blood samples were used to measure blood glucose, lactate, serum insulin, and free fatty acid (FFA), creatine kinase (CK), myoglobin (Mb) and plasma IL-6 concentrations. Serum and plasma samples were obtained by centrifuging at 3000 rpm for 10 min at 4 °C. The plasma and serum samples were stored at −60 °C until analysis. Blood glucose and lactate concentrations were measured using an automatic glucose analyzer (Free style, Nipro Corporation, Osaka, Japan) and lactate analyzer (Lactate pro, Arkray Inc, Kyoto, Japan), respectively. Serum insulin and FFA concentrations were measured using chemiluminescent enzyme immune assays (Fujirebio Inc., Tokyo, Japan) at a clinical laboratory (SRL Inc., Japan). Serum CK and Mb concentrations were also measured at the SRL Clinical Laboratory in Tokyo, Japan. The plasma IL-6 concentration was assayed with an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA). The intra-assay coefficients of variation for each measurement were as follows: 3.1 % for insulin, 1.3 % for FFA, 2.8 % for CK, 2.4 % for Mb and 6.6 % for IL-6.
6
Metabolic Responses to Fasting and Exercise
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Resting blood samples were collected from the antecubital vein on days 1 and 2 following overnight fasting (8:10–8:40) to evaluate blood glucose, lactate, serum leptin, insulin, total ketone body and free testosterone concentrations. Immediately after exercise on day 2, blood samples were further collected to evaluate blood glucose and lactate concentrations. Serum and plasma samples were obtained by 10 min of centrifugation (3000 rpm) at 4°C and stored at -80°C until analysis. Blood glucose and lactate concentrations were evaluated using a glucose analyzer (Free style; Nipro Co., Osaka, Japan) and a lactate analyzer (Lactate Pro; ARKRAY Co., Kyoto, Japan) immediately after blood collection. Serum leptin concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Inc., Minneapolis, MN, USA). The intra-assay coefficient of variation (CV) for the ELISA was 1.1%. Serum insulin, total ketone body and free testosterone concentrations were assayed in a clinical laboratory (SRL, Inc., Tokyo, Japan). The intra-assay CVs were 2.2% for insulin, 2.6% for total ketone body concentration, and 7.1% for free testosterone.
7
Glucose and Insulin Metabolism Assessment
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Blood glucose levels were determined using a blood glucose monitoring system (FreeStyle; Nipro, Osaka, Japan). Serum insulin levels were measured using an ELISA kit (Fujifilm Wako Shibayagi, Gunma, Japan). The homeostatic model assessment of insulin resistance (HOMA‐IR) was calculated by multiplying fasting serum glucose levels by insulin levels. The serum levels of alanine aminotransferase were measured by an enzymatic assay (Fujifilm Wako Pure Chemical Industries, Osaka, Japan). Intraperitoneal glucose tolerance tests (ipGTTs) were performed after fasting for 6 hours, whereas insulin tolerance tests (ITTs) were performed after fasting for 4 hours; the mice were injected intraperitoneally with 3 g/kg body weight D‐glucose or 0.5 U/kg body weight human regular insulin (Humulin R; Eli Lilly, Indianapolis, IN), respectively. Vein blood was collected at 0, 15, 30, 60, and 120 minutes after glucose injection for the ipGTTs and at 0, 15, 30, 45, 60, and 120 minutes after insulin injection for the ITTs. Blood insulin levels were measured at 0, 15, and 30 minutes after glucose injection.
8
Acute Exercise Biomarker Responses
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Blood samples were collected from the antecubital vein before, immediately after, and 60 and 180 min after exercise using serum-separating tubes and tubes containing Na2-ethylenediaminetetraacetic acid (EDTA). Blood samples were centrifuged at 3,000 rpm and 4℃ for 10 min for serum or plasma separation. Obtained serum and plasma were stored at −60℃ until analysis. Blood glucose and lactate concentrations were measured immediately after blood sampling using a glucose analyzer (Free Style; Nipro Corporation, Osaka, Japan) and a lactate analyzer (Lactate Pro; Arkray, Kyoto, Japan), respectively. The concentrations of ferritin, iron, myoglobin (Mb), haptoglobin (Hp), and hepcidin were determined. Serum ferritin, iron, Mb, and Hp concentrations were measured at a clinical laboratory (SRL, Tokyo, Japan). Serum hepcidin concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) with a commercially available kit (R&D Systems, Minneapolis, MN, USA) The intra-assay coefficient of variation (CV) was 1.9, 2.3, 1.6, 2.3, and 2.2%, respectively. From the obtained plasma samples, IL-6 concentration was also measured using ELISA with a commercially available kit (R&D Systems). The intraassay CV values were 2.2, 1.7, and 1.4%, respectively.
9
Metabolic and Inflammatory Response to Exercise
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Venous blood samples were obtained from an antecubital vein before Ex1, immediately after Ex1, immediately before Ex2 (4 h after Ex1), 4 h after Ex2, and 24 h after the onset of Ex1 to determine blood glucose and lactate concentrations. Serum creatine kinase (CK), myoglobin (Mb), leptin, and plasma interleukin-6 (IL-6) concentrations were also evaluated before Ex1 and at 24 h after the onset of Ex1. Serum and plasma samples were obtained by centrifuging for 10 min and were stored at −80 °C until analysis. Serum CK and Mb concentrations were measured at a clinical laboratory (SRL Inc., Tokyo, Japan). The intra-assay CVs were 3.4% for CK and 6.0% for Mb measurements. Serum leptin and plasma IL-6 concentrations were measured with enzyme-linked immunosorbent assay (ELISA) using kits from R&D Systems (Minneapolis, MN, USA). The intra-assay CV was 9.5% for leptin and 8.2% for IL-6, respectively.
The blood glucose and lactate concentrations were measured immediately after blood collection using an automatic glucose analyzer (Free Style, Nipro Corporation, Osaka, Japan) and lactate analyzer (Lactate Pro2; Arkray Inc. Kyoto, Japan), respectively.
The blood glucose and lactate concentrations were measured immediately after blood collection using an automatic glucose analyzer (Free Style, Nipro Corporation, Osaka, Japan) and lactate analyzer (Lactate Pro2; Arkray Inc. Kyoto, Japan), respectively.
10
Exercise-Induced Biomarker Kinetics
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The subjects presented at 8:00. First, a venous blood sample (i.e., pre-exercise sample) was collected after 30 min of rest. The subjects began prescribed exercise at 8:40. Additional blood samples were collected immediately after exercise (9:10), and at 1 h (10:10), 3 h (13:10), and 24 h (09:10) after exercise (Figure 1 ). The collected blood samples were centrifuged for 10 min (3,000 rpm, 4°C), and the serum and plasma samples were stored at −60°C prior to analysis. Blood samples were assessed immediately after collection with automated glucose (Freestyle; Nipro Co., Osaka, Japan) and lactate (Lactate Pro2 LT- 1730; Arkray Inc., Kyoto, Japan) analyzers. Serum cortisol, myoglobin, and CK concentrations were measured at a clinical laboratory (SRL Inc., Tokyo, Japan). Plasma interleukin (IL)-6 and irisin concentrations were determined using enzyme-linked immunosorbent assay (ELISA) kits [HS600B; R&D Systems, Minneapolis, MN, USA (IL-6); EK-067-52 (Lot 605767); Phoenix Pharmaceuticals, Inc., Darmstadt, Germany (irisin)]. All samples for ELISA were assayed in duplicate and the results were averaged. The intra-assay coefficients of variation were: 2.5% for serum cortisol, 2.4% for serum myoglobin, 2.3% for serum CK, 2.5% for plasma IL-6, and 4.3% for plasma irisin.
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