All cells were seeded (5×104 per dish) in a 35-mm dish 2 days before transfection, and the reporter plasmid was transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The appropriate amount of reporter plasmid for each cell line was determined according to differences in transfection efficiency among the cell lines. One day after transfection, cells were treated with 100 nM dexamethasone (Nakalai Tesque, Kyoto, Japan) for 2 h, and the medium was replaced with medium in the absence of phenol red supplemented with 10% FBS and 100 µM D-luciferin (Toyobo). Bioluminescence was measured at 37°C under a 5% CO2 atmosphere and integrated for 1 min at intervals of 10 min using a dish-type luminometer, AB-2550 Kronos Dio (ATTO, Tokyo, Japan) [20] (link), [21] (link). Bioluminescence activity was expressed as relative light units (RLUs). Each experiment was repeated at least four times. The cells were cultured in the luminometer for at least 4 days while the instrument counted their bioluminescence. The obtained crude data (10-min bins) were smoothed by a 10-point moving average method and detrended by subtracting a 12-h moving average from the smoothed data [21] (link).
Dexamethasone (dex)
Manufactured by Nacalai Tesque
Sourced in Japan, United States
Dexamethasone is a synthetic glucocorticoid that is commonly used as a laboratory reagent. It functions as an anti-inflammatory and immunosuppressant compound, and is often utilized in cell culture studies and various biomedical research applications.
Automatically generated - may contain errors
Lab products found in correlation
β glycerophosphate, by Fujifilm (6 mentions)
Picogreen dsdna assay kit, by Thermo Fisher Scientific (5 mentions)
Spectramax m5, by Molecular Devices (5 mentions)
Ascorbic acid, by Nacalai Tesque (4 mentions)
α mem, by Nacalai Tesque (4 mentions)
Indomethacin, by Fujifilm (3 mentions)
Insulin, by Fujifilm (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
β glycerophosphate, by Merck Group (2 mentions)
Insulin, by Nacalai Tesque (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
D luciferin, by Toyobo (1 mentions)
Indomethacin, by Nacalai Tesque (1 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
Triiodothyronine t3, by Merck Group (1 mentions)
3 isobutyl 1 methylxanthine ibmx, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Collagenase type 1, by Worthington (1 mentions)
Dulbecco s modified eagle medium high glucose, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Fujifilm (1 mentions)
22 protocols using dexamethasone (dex)
1
Bioluminescence Assay for Circadian Rhythms
2
Quantifying Osteogenic Differentiation via ALP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate the ALP activity, 4×104 cells were seeded on discs in α-MEM (Nacalai Tesque) containing 10% FBS, antibiotic–antimycotic mix, and the following osteogenic supplements: 10 mM β-glycerophosphate (Wako Pure Chemical Industries, Osaka, Japan), ascorbic acid (Nacalai Tesque), and 10 nM dexamethasone (Nacalai Tesque). The differentiation medium was changed every 3 days. After incubation for 7 or 14 days, the samples were washed with PBS and lyzed with 300 μL of 0.2% Triton X-100, and the lysates were transferred to microcentrifuge tubes. ALP activity was measured using the ALP pNPP Liquid Substrate for enzyme-linked immunosorbent assay (ELISA) Kit (Sigma-Aldrich, St Louis, MO, USA), according to the manufacturer’s protocol. The reaction was terminated by adding 50 μL of 3N NaOH to the 200 μL reaction substrate, and p-nitrophenol production was determined at an optical density of 405 nm using a 96-well microplate reader (SpectraMax® M5; Molecular Devices). The DNA content was determined using the PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. The amount of ALP was normalized to that of DNA within the respective cell lysates.
3
Differentiation of Brown Adipocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The interscapular BAT of Sprague-Dawley rats (4 weeks old, male, Tokyo Laboratory Animals Science Co.) was removed and digested in Hanks’ solution (Worthington Biochemical Co., Lakewood, NJ, USA) containing 3.5% bovine blood albumin (Nacalai Tesque, Kyoto, Japan) and 2 mg/mL of collagenase type I (Worthington Biochemical Co.) for 30 min at 37 °C. Brown progenitor adipocytes were obtained by centrifugation at 1000× g for 5 min. Cells were suspended in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 Ham (DMEM/F-12; Invitrogen, Waltham, MA, USA) containing 10% fetal bovine serum (Invitrogen) at a density of 800,000 cells/mL and cultured in a 35 mm dish. When the cells were 70–80% confluent, the medium was supplemented with 1 nM triiodothyronine (T3: Sigma-Aldrich, St. Louis, MO, USA), 25 μM insulin (Invitrogen), 0.125 mM indomethacin (Nacalai Tesque), 5 μM dexamethasone (Nacalai Tesque), and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX; Invitrogen). After 48 h, the medium was replaced with 1 nM T3 and 25 μM insulin and the culture was continued. As a control, non-induced cells were cultured in DMEM/F-12 without induction of differentiation. After 72 h, cells were treated with isoproterenol or siRNA.
4
Human Bone Marrow-Derived MSC Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human bone marrow-derived MSCs (passage 2) were acquired from PromoCell (Germany) and RIKEN BRC (Japan). In this study, we utilized several lines obtained from 5 different donors (PromoCell: C-12974; RIKEN RBC: MSC-R37, MSC-R43, MSC-R44, and MSC-R50.). The cells were maintained in the basal medium which is low glucose-Dulbecco’s Modified Eagle Medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% antibiotic–antimycotic (Gibco, USA) solution in a humidified incubator at 37℃ under 5% CO2 condition. We carried out the cell passaging every 2–3 days when the cell confluency became 80–90%. For experiments, the cells from passage 4 up to 12 were used. To prepare an osteogenic induction (OI) medium, we utilized high glucose-Dulbecco’s Modified Eagle Medium (Gibco, USA) containing 50 μM ascorbic acid (Wako, Japan), 10 mM β-glycerophosphate (Sigma, USA), and 100 nM dexamethasone (Nacalai Tesque, Japan). To prepare for 2D monolayer samples, 200,000 cells were subcultured on a 35 mm diameter culture dish to become confluent after 2-day incubation. This study was approved by the Ethics Committee of Institute for Frontier Life and Medical Sciences, Kyoto University (Clinical Protocol No. 89).
5
Evaluating Alkaline Phosphatase Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In order to evaluate ALP activity, 4 × 104 cells were seeded on specimens and cultured in α-MEM containing 10% fetal bovine serum, antibiotic-antifungal agent, 10 mM glycerophosphate (Wako Pure Chemical Industries, Osaka, Japan), and 10 nM dexamethasone (Nacalai Tesque). The differentiation medium was changed every 3 days. Following 7 or 14 days of incubation, samples were washed with PBS, and cells that had attached to the sample surface were dissolved with 300 μL of 0.2% Triton X-100. ALP activity was evaluated by an alkaline phosphatase luminometric enzyme-linked immunosorbent assay (ELISA) kit (Sigma-Aldrich) in accordance with the manufacturer’s instructions. A PicoGreen dsDNA analysis kit (Invitrogen/Life Technologies) was utilized to evaluate the DNA content. The amount of ALP was normalized to the amount of DNA in each cell lysate.
6
Adipocyte Differentiation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded at a density of 1 × 105/cm2 and cultured in 10% FBS-DMEM until over-confluent. Subsequently, the cells were cultured in 10% FBS-DMEM supplemented with 10 μg/mL insulin (FUJIFILM Wako Pure Chemical, Osaka, Japan), 200 μM indomethacin (FUJIFILM Wako Pure Chemical), 1 μM dexamethasone (Nacalai Tesque), and 500 μM 3-Isobutyl-1-methylxanthine (FUJIFILM Wako Pure Chemical) for seven days. Cells cultured in 10%FBS-DMEM were used as controls. To detect the formation of lipid vacuoles, the cells were fixed with 4% PFA and stained with Oil Red O solution (0.5% oil red O in 60% isopropanol) (Sigma–Aldrich, St. Louis, MO, USA). The stained dye was eluted with 100% isopropanol and the absorbance was measured at 520 nm.
7
Anti-Inflammatory Mechanisms of Cinnamate-ZLH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RAW 264.7 cell line was sourced from the American Type Culture Collection (ATCC, Manassas, VA, USA), cultured and maintained in Dulbecco’s modified Eagle’s medium, and supplemented with 10% fetal bovine serum and 1% antibiotics. LPS and dexamethasone were sourced from Nacalai Tesque Inc (Tokyo, Japan). CA was supplied by Acros (Geel, Belgium), while ZLH and cinnamate-ZLH (ZCA) were synthesized and characterized in the Institute of Advanced Technology, Universiti Putra Malaysia.39 (link) PGE2 and an enzyme-linked immunosorbent assay kit were purchased from R&D Systems (Minneapolis, MN, USA). All Western blotting apparatus and reagents were from Bio-Rad (St Louis, MO, USA). The primary antibodies iNOS, COX-2, NF-κB, and appropriate secondary antibodies (goat anti-rabbit and goat anti-mouse) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Enhanced chemiluminescence substrates were manufactured by Nacalai Tesque Inc, while the polyvinylidene fluoride membrane was from Bio-Rad.
8
Rat Bone Marrow Mesenchymal Stem Cell Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The animal experiments were conducted in accordance with the Guidelines for Animal Experimentation of Osaka Dental University (approval 13-02039). The rat BMMSCs were obtained from the femurs of 8-week-old Sprague Dawley rats. BMMSCs were maintained in growth medium containing minimal essential medium (Nacalai Tesque Inc., Tokyo, Japan), 10% fetal bovine serum (FBS; Nacalai Tesque Inc.), and antibiotic-antimycotic mixed stock solution (Nacalai Tesque Inc.) and cultured in a humidified atmosphere with 5% CO2 at 37°C. After 3 days, the medium was replaced with the nonadherent cells removed, and thereafter the medium was changed every 3 days. When the culture grew to about 80% confluence, the BMMSCs were trypsinized, using 0.5 g/L trypsin and 0.53 mmol/L ethylenediaminetetraacetic acid (Nacalai Tesque Inc.), and were seeded on specimens at a density of 4 × 104 cells/cm2. The medium was removed and replaced with differentiation medium containing 10% FBS, antibiotic–antimycotic mixed stock solution, and osteogenic supplements: 10 mM β-glycerophosphate (Wako Pure Chemical Industries, Osaka, Japan), ascorbic acid (Nacalai Tesque Inc.), and 10 nM dexamethasone (Nacalai Tesque Inc.). This differentiation medium was changed every 3 days.
9
Adipocyte Differentiation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sorted cells were cultured on a collagen I-coated plate and proliferated in DMEM-10% FBS supplemented with 10 ng/ml murine basic-FGF (Peprotech, Rocky Hill, HJ)22 (link). Twelve to 24 h after reaching 100% confluency, the cells from subcutaneous WAT were treated for 2 days with differentiation medium, 10% FBS-supplemented DMEM containing 1 μM of insulin (Nacalai Tesque, Kyoto, Japan), 0.5 mM 1-methyl-3-isobutyl-xanthine (Nacalai Tesque), and 1 μM dexamethasone (Nacalai Tesque). For the cells from mesenteric WAT, 10 μM pioglitazone, a PPARγ agonist, was added to the differentiation medium because the cells could not otherwise differentiate into adipocytes. To assess in vitro differentiation ability, an equal number of antigen-positive and antigen-negative cells or CFU-Fs was seeded, and the cells were cultured at up to 100% confluency and then treated with differentiation medium within 4 days after seeding. Differentiated cells were further maintained in DMEM-10% FBS. To accurately examine in vitro differentiation ability, the cells of a certain fraction and its counter fraction were cultured in the same way, and induced at the same time.
10
Optimizing CYP3A1 Enzyme Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dulbecco’s modified Eagle’s medium (DMEM)/Nutrient Mixture F-12 Ham, APAP, l -glutamine, l -ascorbic acid, anti-GAPDH antibody and TritonTM X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Collagenase, insulin human recombinant and cycloheximide were purchased from Wako Pure Chemical Industries (Osaka, Japan). Soybean trypsin inhibitor powder, HEPES buffer (1 M) and Dynabeads® Protein G were purchased from Thermo Fisher Scientific (Gibco®; Waltham, MA, USA). 2-Mercaptoethanol, dexamethasone, Nonidet® P-40 and polyoxyethylene sorbitan monolaurate were purchased from Nacalai tesque (Kyoto, Japan). MG132 (Z-Leu-Leu-Leu-H aldehyde) was purchased from Peptide Institute (Osaka, Japan). Proteasome substrates II, III and VI, and Fluorogenic and anti-rabbit CYP3A1 antibodies were purchased from Merck Millipore (Calbiochem®; Darmstadt, Germany). Penicillin G potassium and streptomycin were purchased from Meiji Seika Pharma (Tokyo, Japan). Anti-mouse CYP3A1, anti-rabbit CHIP, anti-rabbit gp78-2, anti-rabbit Nrf2 and anti-mouse Ub antibodies were purchased from Santacruz Biotechnology (Dallas, Texas, USA). P450-GloTM CYP3A assay with luciferin-IPA was purchased from Promega (Fitchburg, WI, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!