RAW 264.7 cells were plated in 96-well plates at a density of 1 × 104 cells/well and incubated overnight at 37 °C under 5% CO2. Then, the cells were treated with compounds 1 –15 and 1a –15a at a concentration of 10 µM, respectively for 1 h and then stimulated with LPS (1 µg/mL). Dexamethasone (Solarbio, D8040, Beijing, China) was used as the positive control. Then, 24 h later, NO production was determined by measuring the nitrite content using Griess reagents. Fifty microliters of culture supernatants were transferred to 96-well plates and mixed with 50 μL Griess reagent A. After incubation at room temperature for 5 min, the samples were mixed with an equal volume Griess reagent B for another 5 min. The absorbance of each mixture at 540 nm was measured using a microplate reader.
D8040
Manufactured by Solarbio
Sourced in China
The D8040 is a laboratory equipment designed for performing fundamental analytical tasks. It is a versatile instrument that can be utilized in various scientific disciplines. The core function of the D8040 is to facilitate precise measurements and data collection for research and analysis purposes.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase, by Merck Group (1 mentions)
Pronase, by Merck Group (1 mentions)
P3376, by Beyotime (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Triiodothyronine t3, by Merck Group (1 mentions)
I8830, by Solarbio (1 mentions)
Fetal bovine serum (fbs), by Solarbio (1 mentions)
I7018, by Merck Group (1 mentions)
3 isobutylmethylxanthine, by Merck Group (1 mentions)
Penicillin streptomycin, by Solarbio (1 mentions)
H8090, by Solarbio (1 mentions)
G8550, by Solarbio (1 mentions)
Ascorbic acid, by Solarbio (1 mentions)
4 protocols using d8040
1
Screening Compounds for Anti-Inflammatory Activity
2
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.
3
Isolation and Differentiation of Murine White Adipose Stromal Vascular Fraction Cells
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Primary white adipose SVF cells were cultured as we described previously (Shan et al., 2016 (link)). Briefly, the inguinal fat pad was collected from 6-week-old female mice and washed with PBS twice. Then, the fat pad was minced with scissors and digested with collagenase type I (1.5 mg/ml, #SCR103, Sigma-Aldrich) at 37°C for 40 min. When the digestion was finished, the growth medium contained 85% high glucose DMEM medium (#11965126, Thermo Fisher Scientific) and 15% fetal bovine serum (#10099141, Thermo Fisher Scientific) was added to dilute the collagenase. The tissue debris was removed through a 70-μm cell strainer. The medium was subjected to centrifuge to get SVF cells pellet. SVF cells were resuspended with the growth medium. When the cells reached 90% confluence, they were induced to adipogenesis, with a cocktail containing DMEM, 10% fetal bovine serum, 2.85 mM recombinant human insulin (#I8830, Solarbio), 0.3 mM dexamethasone (#D8040, Solarbio), and 0.63 mM 3-isobutyl-methylxanthine (#I7018, Sigma-Aldrich). After 4 days, the cocktail was switched to a DMEM medium supplemented with 10% fetal bovine serum, 10 nM triiodothyronine (T3, #T6397, Sigma-Aldrich), and 200 nM insulin to induce mature adipocytes.
4
Osteogenic Differentiation of hBMSCs from Femoral Head Necrosis Patients
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The hBMSCs from patients with femoral head necrosis secondary to senile femoral neck fractures were selected as the experimental subjects. Cells were seeded on 6-well plates. When cells reached 60–70% confluence, the medium was changed with the osteogenic differentiation medium to induce differentiation. The osteogenic differentiation medium was freshly prepared before use, which contained basic medium, 10% fetal bovine serum, 10 nmol/L dexamethasone (D8040; Beijing Solarbio Co., Ltd., China), 10 nmol/L ascorbic acid (A8100; Beijing Solarbio Co., Ltd., China), 50 mol/mL of glycerol phosphatide (Sigma-Aldrich, USA), 1% penicillin–streptomycin and 1% HEPES (H8090; Beijing Solarbio Co., Ltd., China). After 21 days of osteogenic differentiation, hBMSCs in the 6-well plate were washed with PBS and fixed with neutral formalin for 30 min. The fixation fluid was discarded, and the cells were rewashed with PBS. One milliliter of ARS agent (G8550; Beijing Solarbio Co., Ltd., China) was added to the wells for staining for 5 min. Then cells were washed and dried. The staining effect was observed under a microscope (DYF-880; Shanghai Dianying Optical Instrument Co., Ltd., China).
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