HepG2-hNTCP-C4 cells were cultured in collagen coated flasks (0.1 mg/mL collagen [Sigma, C3867]) as described earlier at 37 °C in a humidified incubator containing 5% CO2. The growth media consisted of HEPES buffered DMEM (Invitrogen, 2320032) supplemented with 200 units/ml penicillin, 200 μg/ml streptomycin (Gibco, 15140-122), 10% FBS (Gibco, 16000-044), 50 μM hydrocortisone (Himedia, TC344), 5 μg/ml insulin (Himedia, TC190) and 400 μg/ml G418 (InvivoGen, ant-gn-1).
Insulin
Manufactured by HiMedia
Sourced in India
Insulin is a hormone produced by the pancreas that regulates blood sugar levels. It is a core component in the treatment of diabetes, a condition characterized by the body's inability to effectively use or produce insulin.
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Lab products found in correlation
Dulbecco s modified eagle s medium dmem, by HiMedia (2 mentions)
Hepes buffered dmem, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Eugenol, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Oil red o, by HiMedia (1 mentions)
Dexamethasone (dex), by HiMedia (1 mentions)
Formalin, by HiMedia (1 mentions)
Isopropanol, by HiMedia (1 mentions)
Pioglitazone hydrochloride, by Merck Group (1 mentions)
Metformin hydrochloride, by Merck Group (1 mentions)
Ix73 inverted microscope, by Olympus (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Prolactin, by Merck Group (1 mentions)
Dimethyl sulphoxide, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by HiMedia (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by HiMedia (1 mentions)
Thiazolyl blue tetrazolium bromide mtt, by HiMedia (1 mentions)
6 protocols using insulin
1
Cultivation of HepG2-hNTCP-C4 Cells
2
Eugenol-Protein Interaction Characterization
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Eugenol (from natural source with purity of ≥98.5%) and BSA were procured from Sigma-Aldrich. Insulin and lysozyme proteins were obtained either from HIMEDIA (India). All other reagents and chemicals used in this work were purchased either from HIMEDIA or Sigma-Aldrich. Extinction coefficients used for proteins were as follows: 43824 M−1 cm−1 at 280 nm for BSA and 6080 M−1 cm−1 at 278 nm for Insulin. Eugenol stock solution was prepared by dissolving it in ~70% ethyl alcohol solution.
3
Evaluation of Lentinula edodes Powder
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Dried L. edodes was procured from local market in Mumbai, India. The mushrooms were ground and passed through sieve no. 45 to obtain a fine powder. The powder was stored in an airtight container. LC-MS-grade methanol and acetonitrile were procured from Thomas Bakers Pvt. Ltd., Mumbai, India and S.D. Fine Chemicals Ltd, Mumbai, India. The 3T3-L1 adipocytes were procured from NCCS, Pune, India (Request Number: 868/2021-22). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), antibiotic (penicillin-streptomycin solution), Oil Red O, formalin, dexamethasone, insulin, 3-isobutyl-1-methylxanthine (IBMX), and isopropanol were provided by HiMedia, India. Other chemicals used were of analytical grade and obtained from S.D. Fine Chemicals Ltd, Mumbai, India.
4
Adipocyte Response to Anti-diabetic Drugs
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The cultured mature adipocytes were further treated with glucose (5–20 mM) in triplicate along with variable concentration of anti-diabetic drugs (metformin hydrochloride (50–200 mg/mL), pioglitazone hydrochloride (5–20 mg/mL) (Sigma Aldrich, USA) and insulin (10–25 mg/mL) (HiMedia, India)). The level of TNF-α was measured by ELISA. The cells were firstly centrifuged at 300×g for 2 min, and thereafter supernatant was used for assay for its estimation using a commercially available kit from Invitrogen (USA) according to the manufacturer’s instructions.
5
Goat Mammary Epithelial Cell Differentiation
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Goat mammary epithelial cells at passage 2 were induced to differentiation towards milk-secreting cells [44 (link)]. In addition to normal constituents of growth media, induction media was supplemented with the combination of 5 µg/ml prolactin (Sigma-Aldrich), 5 μg/mL insulin (Himedia), 5 μg/mL hydrocortisone and 20 ng/ml EGF (Sigma-Aldrich) to induce the expression of CSN-2. Culturing the cells in induction media for 5 days induced differentiation. After 5 days the RNA was isolated from cells, cDNA was synthesised and gene expression analysis of CSN-2 was done by RT-qPCR. Prior to RNA isolation, the differentiated cells were visualized under an Inverted Microscope (IX73, Olympus, Japan).
6
Adipogenic Differentiation Assay Protocol
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Syringic acid (SA, Sigma), Dulbecco's Modified Eagle's Medium (DMEM, HiMedia), phenol red-free Dulbecco's modified eagle's medium (HiMedia), insulin (HiMedia), dexamethasone (DEX, HiMedia), 0.25% trypsin-EDTA (HiMedia), Dulbecco's Phosphate Buffered Saline (DPBS, HiMedia), 3-isobutyl-1-methylxanthine (IBMX, Sigma), dimethyl sulphoxide (DMSO), free glycerol reagent (Sigma), foetal bovine serum (FBS, Gibco®), 100X antibiotic-antimycotic (Gibco®), thiazolyl blue tetrazolium bromide (MTT, HiMedia), β-nicotinamide adenine dinucleotide (reduced) disodium salt (β-NADH, SRL Pvt. Ltd), Oil-Red-O (ORO, HiMedia), nitroblue tetrazolium chloride (NBT, HiMedia), fatty acid-free bovine serum albumin (BSA, HiMedia) . All reagents and chemicals used were of tissue culture grade.
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