Sixth instar 6 h larvae were subjected to 20E (H5142, Sigma Aldrich, USA; 200, and 500 ng/larva) or human insulin (P3376, Beyotime Biotechnology, 1, 5, and 10 μg/larva) injection with an equivalent amount of dimethyl sulfoxide (DMSO) or PBS injected for controls. Five microliters of solution were injected into the hemolymph of the sixth instar 6 h larvae, individually. The larvae were sacrificed at 1, 3, and 6 h after injection of different concentrations of 20E or insulin. Tissues were dissected from the sacrificed larvae for qRT-PCR (Quantitative real-time PCR) analysis or western blot. Five animals were used for per replicate and more than three biological experiments were conducted for each group.
P3376
Manufactured by Beyotime
Sourced in United States, China
The P3376 is a precision laboratory instrument designed for measuring and monitoring various parameters in controlled environments. It is a versatile tool commonly used in research and testing applications. The core function of the P3376 is to provide accurate and reliable data collection, without further interpretation or extrapolation on its intended use.
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6 protocols using p3376
1
Hormone Regulation in Insect Larvae
2
Quantifying Liver Tissue and Plasma Biomarkers
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Serum insulin (P3376, Beyotime Biotechnology), TNFα (PT512, Beyotime Biotechnology), IL-6 (PI326, Beyotime Biotechnology), and PDGF-AA (SEA523Mu, Cloud-Clone Corp., Wuhan, China) concentrations in the liver tissue and plasma were quantified using mouse ELISA kits according to the manufacturer’s instructions. The levels of secreted PDGF-AA in the cell culture supernatants were determined using a human ELISA kit (SEA523Hu, Cloud-Clone Corp). Active RhoA was detected in a colorimetric RhoA activation assay (G-LISA) (BK124, Cytoskeleton, Denver, CO, USA).
3
Metabolic assessment in NMN mice
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Analyses of fasted glucose, intraperitoneal glucose tolerance testing (IPGTT), and insulin tolerance testing (ITT) levels were conducted only in and NMN-BS groups at the end of 18-wk NMN supplementation. Fasted glucose levels were detected in tail-vein blood collected from mice in the control ( ) and NMN-treated groups ( /group) using a JPS-7 glucometer (Yicheng). For IPGTT, the mice were injected intraperitoneally (IP) with 20% glucose solution (wt/vol; dissolved in PBS) at a dose of ( /group) after being fasted for 15 h. Peripheral blood samples were collected for glucose detections at 15, 30, 60, 90, and 120 min, respectively, post injection. ITT measurement was performed in the two groups of mice following 4 h fasting and IP injection of insulin (P3376; Beyotime Biotechnology). The blood glucose levels were measured subsequently at 15, 30, 60, and 90 min, respectively, post injection ( /group). The test values of IPGTT and ITT at each time point were normalized to that at the initial time point (percentage blood glucose relative to initial) and areas under the curve were calculated.
4
Isolation and Culture of Primary Hepatocytes from WT and L-KO Mice
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Primary hepatocytes were isolated and cultured from the livers of WT and L-KO mice fed the HFCD-HF/G as described previously69 (link). Briefly, following anesthesia, the inferior vena cava was cannulated and the liver was perfused in situ with 40 mL pre-warmed EGTA solution and 40 mL solution containing 0.35 mg/mL pronase (no. P5147, Sigma-Aldrich, St. Louis, MO, USA), followed by 40 mL solution containing 0.55 mg/mL collagenase (no. V900893, Sigma-Aldrich). After perfusion, the liver was crushed, and hepatocytes were released into the DMEM. The cell suspension was filtered through a 100 μm cell strainer and centrifuged at 50 ×g at 25 °C for 3 min. After washing three times, the cells were suspended in DMEM supplemented with 10 mM glucose, 10% fetal bovine serum, 100 nM insulin (P3376, Beyotime Biotechnology, Shanghai, China), and 100 nM dexamethasone (D8040, Solarbio Life Sciences, Beijing, China), and then plated on 60 mm diameter plastic plates69 (link). After cell attachment, the medium was replaced with serum-free media, and the cells were used for experiments on the following day.
5
Glucose and Insulin Tolerance Tests
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For GTT, after a 12-h fast, basal blood glucose levels of mice in each group were measured by cutting tail veins. Then the mice were administered glucose solution at 1.5 g kg−1 body weight by intraperitoneal injection, and blood glucose levels were measured at 15, 30, 60, and 120 min after glucose injection. For ITT, after a 4-h fast, we measured the basal blood glucose of each group of mice. Then the mice were given an intraperitoneal injection of insulin (#P3376, Beyotime) (1.0 and 1.5 IU kg−1 body weight for NCD-fed and HFD-fed mice, respectively). The blood glucose levels were measured at 15, 30, 60, and 120 min after insulin administration.
6
Insulin and 20E Regulation of Larval Physiology
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Different concentrations (1, 2, 5, and 10 μg/larva) of human insulin (P3376, Beyotime Biotechnology) or 20E (50, 100, 200, and 500 ng/larva; H5142, Sigma- Aldrich, St. Louis, MO, USA) diluted with PBS and then 5 μL of insulin or 20E were injected into the hemolymph of the 6th-6 h larva for 6 h, individually. Equally diluted PBS for insulin and DMSO for 20E were used as controls. Insulin at 5 μg/larva or 20E at 500 ng/larva was injected into the hemolymph of the 6th-6 h larva from 1 h to 24 h; PBS and DMSO were used as controls. Each treatment used three larvae, from which the fat body was extracted using 40 mM Tris-HCl for western blotting.
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