In vitro glucose production assay was performed as follows, based on a previous study.27 (link) Isolated hepatocytes were placed in glucose production buffer containing 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.2 mM CaCl2, 20 mM NaHCO3, 25 mM HEPES pH 7.4, 0.025% BSA and 0.1mM 8-(4Chlorophenylthio) denosine 3′,5′-cyclic monophosphate sodium (pCPT-cAMP; Sigma, USA; #C3912). The buffer contains 0.4mM pyruvate +4.0mM lactate, 4mM glutamine, 4mM alanine or 0.5mM glycerol, unless otherwise indicated. BCKAs or 5μM UK5099 (sigma, USA; # PZ0160) was also added in the buffer when indicated. After incubated in the buffer for 5 h, conditioned medium was removed for glucose measurement and the hepatocytes were lysed with RIPA buffer. Glucose levels were measured by colorimetric assay (AUTOKIT Glucose, WAKO, Japan; # NC9927772) and the protein concentration was determined by Pierce BCA Protein Assay Kit (Thermo, USA; # 23227). For HepG2, 5μM dexamethasone (WAKO, Japan; #047–18863) was added to the medium over-night before the assay and 2mM pyruvate +20mM lactate + 5μM dexamethasone were added in the assay buffer, based on a previous study.44 (link)
Dexamethasone (dex)
Manufactured by Fujifilm
Sourced in Japan, United States
Dexamethasone is a synthetic corticosteroid medication used in laboratory settings. It functions as a potent anti-inflammatory agent, with the primary purpose of reducing inflammation and suppressing immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (20 mentions)
Insulin, by Fujifilm (13 mentions)
Oil red o, by Merck Group (10 mentions)
3 isobutyl 1 methylxanthine, by Fujifilm (8 mentions)
Dexamethasone (dex), by Merck Group (8 mentions)
β glycerophosphate, by Fujifilm (7 mentions)
Insulin, by Merck Group (6 mentions)
Recombinant human insulin, by Fujifilm (6 mentions)
Indomethacin, by Fujifilm (5 mentions)
Ascorbic acid 2 phosphate, by Fujifilm (5 mentions)
L ascorbic acid, by Fujifilm (5 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (5 mentions)
β glycerophosphate, by Merck Group (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
L ascorbic acid 2 phosphate, by Fujifilm (4 mentions)
Isobutylmethylxanthine, by Merck Group (4 mentions)
Lipopolysaccharides (lps), by Merck Group (4 mentions)
α mem, by Fujifilm (4 mentions)
Penicillin, by Nacalai Tesque (4 mentions)
Ascorbic acid, by Fujifilm (4 mentions)
118 protocols using dexamethasone (dex)
1
In Vitro Hepatocyte Glucose Production
2
Circadian Rhythm Synchronization Protocols
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The circadian rhythm was synchronized via stimulation of 0.1 µM Dex (Wako, Osaka, Japan) for 2 h or 10 µM Frk (Wako, Osaka, Japan) for 1 h. After each stimulation, the medium was replaced with a standard medium (referred as 0 h), and RNA extraction was performed every 4 h. The initial induction of PER1 was examined by adding 0.1 µM Dex or 10 µM Frk. RNA extraction was performed every 30 min, while the time of addition was 0 min. The simulated body temperature rhythm was achieved by using a 33 °C condition for 12 h and a 37 °C condition for 12 h, for a total of 5 days, and then the cells were cultured for another 24 h at 37 °C. RNA extraction was performed every 4 h from day 5 to day 6. Gene knockdown was performed using Dharmacon™ ON-TARGETplus SMARTpool™ siRNA (Horizon Discovery, Lafayette, CO, USA), which consists of a mixture of four siRNAs for same target gene. Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) was used to add a final concentration of 20 nM siRNA. siRNA was added on day 4 of the temperature rhythm, and removed 24 h later. Hypoxic treatment was performed by culturing cells under a 1% O2 condition for the indicated periods in a multi-gas incubator (APM-30DR; ASTEC, Fukuoka, Japan).
3
Osteogenic Differentiation of hDPSCs
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hDPSCs (5 × 104 cells) were cultured in an osteogenic differentiation medium containing l -ascorbic acid-2-phosphate (0.2 mM; Fujifilm Wako Pure Chemical), beta-glycerophosphate (5 mM; Fujifilm Wako Pure Chemical), dexamethasone (1 nM; Fujifilm Wako Pure Chemical), and BMP-2 (100 ng/ml; Fujifilm Wako Pure Chemical) for 21 days. Mineralized nodules were stained with Alizarin Red S (Fujifilm Wako Pure Chemical).
4
Osteogenic Differentiation of Dental Pulp Stem Cells
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Dental pulp stem cells were cultured in 12-well plates containing osteogenic differentiation media with and without TH for 14 days. We used αMEM supplemented with 10% FBS, 1% P/S, and 10 nM dexamethasone (Wako Pure Chemical Industries) for osteogenic induction (osteogenic medium (OM)). We added TH (Takeda Chemical Industries) at the optimal concentration of 10−6 M to the OM, as described previously [15 (link)].
5
Zebrafish Xenograft Imaging Protocol
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For the first study (Fig. 7 Study 1) the zebrafish were immersed in Dexamethasone (FUJIFILM Wako Pure Chemical Corporation, Japan, 10 g/ml) for 2 days, a drug to suppress the immune system49 (link). All animals were kept separately in six-well plates. Four zebrafish were injected with MCF-7 cells (concentration: cells in 1 ml PBS) and in two control animals only the PBS solution was injected at the same location, see Fig. 7 . After 7 days post-injection (dpi) all fish were imaged under anesthesia (Tricaine (0.16 mg/ml)) using the JM-OCT prototype. The fish were placed in a petri dish and each imaging session took less than 5 min, to ensure the survival of the animals. All zebrafish were sacrificed after OCT imaging with an overdose of Tricaine and fixated in 4% paraformaldehyde (FUJIFILM Wako Pure Chemical Corporation, Japan) for histological workup.
6
Chondrogenic Induction of Cells
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Two-dimensional chondrogenic induction was performed as previously described [32 (link)]. Briefly, cells (1.5 x105) were suspended in 5 μl of chondrogenic medium (DMEM/F12 (Life Technologies), 1% (v/v) ITS1 mix (BD), 0.17 mM AA2P (Sigma), 0.35 mM Proline (Sigma), 0.1 μM dexamethasone (WAKO), 0.15% (v/v) glucose (Sigma), 1 mM Na-pyruvate (Sigma), and 2 mM GlutaMax (Life Technologies) supplemented with 40 ng/ml PDGF-BB (R&D System) and 1% (v/v) FBS (Nichirei, Inc., Tokyo, Japan)). They were subsequently transferred to fibronectin-coated 24-well plates (Corning, Inc., NY, USA). One milliliter of the chondrogenic medium was added after 1 h. TGFb3 (R&D System) was subsequently added at 10 ng/ml on days 6 to 10. Differentiation was confirmed on day 10 using Alcian Blue staining.
7
Adipocyte Differentiation Induction Protocol
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D-MEM/Ham’s F-12, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX) and insulin were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Resveratrol was a product of Tokyo Chemical Industry (Tokyo). Newborn calf serum (NBCS) and fetal bovine serum (FBS) were purchased from Thermo Fisher (Waltham, MA) and Biosera (Boussens, France), respectively. SB203580 was purchased from Merck Millipore (Darmstadt, Germany). Oil Red O and anti-β-Actin antibody were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies against C/EBPβ (H-7) and C/EBPδ (C-6) were products of Santa Cruz Biotechnology (Dallas, TX). Antibodies against p38 MAPK (D13E1), phospho-p38 MAPK (Thr180/Tyr182) (D3F9), ERK1/2 (137F5) and phospho-ERK1/2 (Thr202/Tyr204) (D13.14.4E) were purchased from Cell Signaling Technology (Danvers, MA). Oligonucleotides were supplied by FASMAC (Kanagawa, Japan).
3T3-L1 cells were supplied by the Riken Cell Bank (Tsukuba, Japan) and cultured with D-MEM/Ham’s F-12 medium supplemented with 10% heat-inactivated NBCS at 37 °C under humidified 5% CO2.
3T3-L1 cells were supplied by the Riken Cell Bank (Tsukuba, Japan) and cultured with D-MEM/Ham’s F-12 medium supplemented with 10% heat-inactivated NBCS at 37 °C under humidified 5% CO2.
8
Chondrogenic Differentiation of Human Bone Marrow MSCs
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Human bone marrow MSC were obtained from five donors (41–65 years old) undergoing treatment with intra-articular injections of autologous MSC at Avenue Cell Clinic in accordance with a protocol approved by the Institutional Ethics Committee. Human MSC around passage 4–5 were induced to undergo chondrogenic differentiation. For the pellet culture, 2 × 105 cells were placed in 96 deep well polypropylene plates and centrifuged at 500 × g for 5 minutes with basal media; DMEM/HG supplemented with 1% ITS premix (Corning; Corning, NY), 50 µg/mL 2-Phospho-L-ascorbic acid trisodium, 40 µg/mL L-proline (Sigma-Aldrich), 1% PS. The next day, the media was changed to chondrogenic-induction media; basal media supplemented with 10 nM dexamethasone (Wako), 10 ng/mL recombinant human transforming growth factor-β1 (rhTGF-β) (Oriental Yeast Co) and 100 ng/mL recombinant human bone morphogenetic protein-2 (rhBMP-2) in the presence or absence of KAG-308. The media and KAG-308 were changed twice a week. After 21 days, the pellets were analyzed.
9
3T3-L1 Cell Differentiation and Methylation Inhibition
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3T3-L1 pre-adipocytes, kindly provided by Dr. Hosaka at Kyorin University, Tokyo, Japan, were grown to confluence at 37°C and 7.5% CO2/air in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma, MO, USA) supplemented with 10% calf serum (Thermo Fisher Scientific, MA, USA). Cells were differentiated two days after reaching confluence by replacing the media with DMEM containing 10% fetal bovine serum (GE Healthcare, Little Chalfont, UK), 10 μg/mL insulin (Wako, Tokyo, Japan), 500 μM isobutylmethylxanthine (Sigma, MO, USA), 1 μM dexamethasone (Wako, Tokyo, Japan), and 1μM troglitazone (Cayman Chemical, MI, USA). After another two days, media were replaced with 10% fetal bovine serum in DMEM, and refreshed daily thereafter. To inhibit methylation, 3T3-L1 pre-adipocytes were cultured for seven days in DMEM supplemented with 10% calf serum and 0.5–5 μM 5-azacytidine (Sigma, MO, USA). Media were replaced every other day and cells were subsequently differentiated and maintained as described. Cells were harvested at day 0 and day 6–8.
10
Expansion and Hepatic Maturation of hiPSC-Derived Liver Progenitor Cells
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To expand hiPSC-derived LPCs, we cultured the cells on mitomycin C-treated (Wako) HUVEC/MSC or hiPSC-derived NPC feeder cells (50,000 cells/cm2) in DMEM/F12 (Sigma-Aldrich) supplemented with 10% fetal bovine serum (JRH Biosciences), penicillin-streptomycin-glutamine, insulin-transferrin-selenium, N2 supplement, MEM non-essential amino acids solution, L-glutamine (Life Technologies), ascorbic acid (1 mM), nicotinamide (10 mM), N-acetylcysteine (0.2 mM) (Sigma-Aldrich), dexamethasone (1 × 10−7 M), Y27632 (5 μM) (Wako), and A83-01 (2.5 μM) (Tocris) for 14 days. To induce hepatic maturation of hiPSC-derived LPCs, we cultured cells in HBM (Lonza) supplemented with HCM SingleQuots (excluding epidermal growth factor) and oncostatin M (20 ng/mL) (PeproTech) for 5 days.
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