Saikosaponin D (purity >98%) was purchased from MCE (New Jersey, United States). DMSO (Meilunbio, Dalian, China) was used to dissolve Saikosaponin D and diluted in the cell culture until DMSO <0.1%. Recombinant mouse IL-1β was obtained from Peprotech (United States). Safranin-O and Fast Green and tartrate-resistant acid phosphatase staining (TRAP) were acquired from Solarbio (Beijing, China). Primary antibodies were applied in this study: antibodies against P65 (#8242), p-P65 (#3033), IκBα (#4814), p-IκBα (#2859), ERK (#4695), p-ERK (#4370), JNK (#9252), p-JNK (#9255), P38 (#8690), and p-P38 (#4511S) were acquired from CST (1:1,000, Cambridge, MA, United States). Anti-iNOS (1:1,000, ab178945), anti-COX2 (1:4,000, ab179800), anti-Aggrecan (1:1,000, ab3778), and anti-ADAMTS5 (1:250, ab41037) were purchased from Abcam (Cambridge, United Kingdom). Antibodies against MMP13 (1:2000, 18165-1-AP), Collagen II (1:1,000, 28459-1-AP), Nrf2 (1:2000, 66504-1-Ig), HO-1 (1:1,500, 27282-1-AP), Lamin B (1:10000, 66095-1-Ig), and Actin β (1:8,000, 66009-1-Ig) were obtained from Proteintech (Wuhan, China). Goat anti-mouse and goat anti-rabbit were acquired from Thermo Fisher (United States).
Dimethyl sulfoxide (dmso)
Manufactured by Meilun
Sourced in China
DMSO is a colorless, odorless, and polar aprotic solvent used in various laboratory applications. It has a high boiling point and is miscible with water and many organic solvents. DMSO is commonly used as a cryoprotectant and in the preparation of samples for spectroscopic analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
12 well transwell plate, by Corning (2 mentions)
Non essential amino acids solution, by Merck Group (2 mentions)
Sodium pyruvate, by Solarbio (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Chloroquine, by Merck Group (1 mentions)
Acridine orange, by Merck Group (1 mentions)
Pepstain a, by Merck Group (1 mentions)
Mg132, by Meilun (1 mentions)
Cleaved parp antibody, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
P53 sc 126, by Santa Cruz Biotechnology (1 mentions)
Etoposide, by Meilun (1 mentions)
Ab179800, by Abcam (1 mentions)
Ab41037, by Abcam (1 mentions)
Ab3778, by Abcam (1 mentions)
Ab178945, by Abcam (1 mentions)
Safranin o, by Solarbio (1 mentions)
Recombinant mouse il 1β, by Thermo Fisher Scientific (1 mentions)
10 protocols using dimethyl sulfoxide (dmso)
1
Saikosaponin D Pharmacological Evaluation
2
Autophagy Modulation in Cell Biology
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BA (Meilunbio, Dalian, China; MB5971), Chloroquine (Sigma, St. Louis, MO, USA; C6628), E-64d (Sigma, E8640) and Pepstain A (Sigma, P4265) were dissolved in DMSO (Sigma, D2650). Acridine orange (Sigma, A6014) and 3-MA (Meilunbio, MB5063) were dissolved in PBS. MG132 (Meilunbio, MB5137), Etoposide (Meilunbio, MB1102-S) and CHX (Meilunbio, MB2208) were dissolved in DMSO. LC3 (MBL, Beijing, China; PM036) was bought for immunoblot or immunofluorescence analysis. Antibodies including AKT, phosphorylation of AKT (Ser473; 4060), MTOR, phosphorylation of MTOR (Ser2443; 5535), PARP (116 and 89 kd; 9542S), BAX (5023), phosphorylation of p53 (Ser15; 9286) and GAPDH (Santa Cruz Biotechnology(CA, USA) 5174) were purchased from CST (Beverly, MA, USA). Cleaved-PARP antibody was purchased from Sigma (SAB4500487). p53 (SC-126) and LAMP1 (SC-5570) were purchased from Santa Cruz Biotechnology (CA, USA) for immunoblot or immunofluorescence analyses.
3
Quantification of Drug Permeability
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Benzaldehyde (BEN > 99%) was purchased from Chroma Biotechnology Co., Md (Chengdu, China). Parahydroxybenzoic acid (>98%), benzaldehyde (BEN > 99%), acyclovir (ACV > 99%), carbamazepine (CBZ > 99%), vinblastine (VIN > 99%), hydrochlorothiazide (HTZ >99%), and propranolol hydrochloride (PRO >99%) were purchased from Chengdu Alfa Biotechnology Co., Ltd. (Chengdu, China). TMA-DPH was purchased from MedChemExpress (MA, United States). Acetonitrile and formic acid (HPLC grade) were bought from Fisher Chemical (MA, United States). HBSS and MEM were bought from Hyclone (MA, United States), nonessential amino acids solution was purchased from Sigma-Aldrich (Shanghai, China), and sodium pyruvate was bought from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). FBS was purchased from Every Green Biotechnology Co., Ltd. (Zhejiang, China). 0.25% Trypsin–EDTA and penicillin–streptomycin solution was provided by Gibco (NY, United States). DMSO was obtained from Meilunbio (DaLian, China). A 12-well Transwell® plate was purchased from Corning (NY, United States). All other solvents were of analytical agents and aqueous solutions were prepared by double-distilled water.
4
Optimizing Transwell Assay for Cell Culture
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EDM (EDM >99%) and DEDM (DEDM >99%) were purchased from Chengdu Alfa Biotechnology Co., Ltd. (Chengdu, China). Acetonitrile (HPLC grade) was got from fisher chemical (Massachusetts, United States). Non-essential amino acids solution from Sigma-Aldrich (Shanghai, China) and Hank’s balanced salt solution (HBSS), penicillin-streptomycin solution (×100) and MEM were purchased from Hyclone (Massachusetts, United States). Sodium pyruvate was bought from solarbio science and technology Co., Ltd., (Beijing, China). Gibco (New York State, United States) supplied the 0.25% Trypsin-EDTA and penicillin-streptomycin solution. From Every Green Biotechnology Co., Ltd., (Zhejiang, China), FBS was bought. DMSO was obtained from Meilunbio (Dalian, China). 12-well Transwell® plate was acquired from Corning (New York State, United States). The preparation of aqueous solutions uses double-distilled water and all additional solvents are analytical agents.
5
In-House Synthesis and Characterization of ARV-471
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ARV-471 was synthesized in-house, and its identity and purity were assessed using nuclear magnetic resonance spectroscopy, mass spectrometry, and high-performance liquid chromatography. Dog liver microsome (DLM) was purchased from Corning Gentest (Glendale, AZ, USA). 7-Ethoxycoumarin (7-EC) was purchased from J&K Scientific Ltd. (Shanghai, China). Phosphate buffer saline (PBS), MgCl 2 , DMSO, alamethicin, and uridine-5'-diphosphoglucuronic acid (UDPGA) were from Meilunbio (Dalian, China). Nicotinamide adenine dinucleotide phosphate (NADPH) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.
(Shanghai, China). Ammonium acetate was from Sinopharm Group Co., Ltd.
(Shanghai, China). Formic acid was purchased from RHAWN (Shanghai, China).
Acetonitrile (ACN) of LC-MS grade was obtained from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water was freshly prepared with a Millipore purification system (Molsheim, France).
(Shanghai, China). Ammonium acetate was from Sinopharm Group Co., Ltd.
(Shanghai, China). Formic acid was purchased from RHAWN (Shanghai, China).
Acetonitrile (ACN) of LC-MS grade was obtained from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water was freshly prepared with a Millipore purification system (Molsheim, France).
6
Inhibiting Platelet Aggregation and Metastasis
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For inhibiting platelet aggregation, aspirin (catalog no. J20171021, Bayer) at 30 mg/kg or warfarin (catalog no. H31022123, SINE) at 30 mg/kg was given orally once per day to each mouse for 10 days. Distilled water was used as a control. For metastasis inhibition, a selective GRP75 inhibitor MKT-077 (catalog no. A12388, AdooQ Bioscience) at 15 mg/kg was intraperitoneally injected once per day into each mouse. DMSO (catalog no. MB2505, Meilunbio) was used as a control. For in vitro platelet activation, freshly isolated mouse or human platelets were treated with ADP (catalog no. MB1706, Meilunbio), collagen (catalog no. MB5213, Meilunbio), thrombin (catalog no. MB1368, Meilunbio), ionomycin (catalog no. MB7511, Meilunbio), and arachidonic acid (AA, catalog no. MB6012, Meilunbio). In some experiments, a GRP75 inhibitor MKT-077 (catalog no. A12388, AdooQ Bioscience) at 20 mmol/L was pretreated for 30 minutes prior to agonist stimulation.
7
Didymin Antioxidant Assay Protocol
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Didymin (C28H34O14; >98% pure, HPLC-grade) and dimethyl sulfoxide (DMSO) were taken from Dalian Meilun Biological Technology Co., Ltd. (Dalian, China). Juglone, levamisole and dichlorofluorescin diacetate (DCFH-DA) were bought from TransGen Biotech Co., Ltd. (Beijing, China). The superoxide dismutase (SOD) Activity Detection Kit was obtained from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). The Easypure RNA Kit, TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix, and TransStart Green qPCR SuperMix were purchased from TransGen Biotech Co., Ltd. (Beijing, China). All other chemicals were of analytical or reagent grade.
8
Osteoclastogenesis Regulation by SSD
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SSD with >98% purity was obtained from MedChemExpress (NJ, USA). Dimethyl sulfoxide (DMSO) (Meilunbio, Dalian, China) was used to dissolve SSD at a concentration of 1 mmol/L and was stored at −20°C. The culture medium was used for further dilutions in the in vitro experiments, and phosphate-buffered saline (PBS) was used for in vivo experiments. Alpha-modified Eagle medium (α-MEM) was provided by HyClone Laboratories (Logan, UT, USA). Foetal bovine serum (FBS) was obtained from Gibco-BRL (Gaithersburg, USA). Cell counting kit-8 (CCK-8) was obtained from Beyotime (Shanghai, China). M‐CSF (mouse recombinant) and RANKL were provided by R&D Systems (Minneapolis, USA). Primary antibodies, including β‐actin (#4970), P65 (#8242), p-P65 (#3033), P38 (#8690), p-P38 (#4511S), AKT (#4691), p-AKT(#4060), ERK(#4695), p-ERK(#4370), IκBα(#4814), p-IκBα (#2859), PI3K(#4295), p-PI3K (#17366), JNK (#9252) and p-JNK(#9255), were provided by Cell Signaling Technology (1:1000, Danvers, USA). In addition, c-Fos(ab222699) and NFATc1(ab2722) were obtained from Abcam (1:1000, Cambridge, UK), and the dilution of the primary antibodies was purchased from Beyotime Biotechnology.
9
Cytotoxicity Evaluation of Fetal Bovine Serum Compounds
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Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific (Shanghai, China). ISO was procured from Sigma Industry Co., Ltd. (St Louis, MO, USA). In addition, dual anti-sterilization solution (penicillin/streptomycin solution), 0.25% trypsin-EDTA, phosphate-buffered saline (PBS), and Dulbecco’s modified Eagle’s medium (DMEM) were all purchased from Meilun Biotech (Dalian, China). MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) was purchased from Beyotime Biotechnology (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from Meilun Biotech (Dalian, China).
Antibody against β-actin was purchased from Servicebio (Wuhan, China), and the caspase-3, caspase-1, PARP, and cleaved PARP antibodies were obtained from Biosystems Dogs (Shenyang, China). Other antibodies were all purchased from Beyotime Biotechnology (Shanghai, China).
Antibody against β-actin was purchased from Servicebio (Wuhan, China), and the caspase-3, caspase-1, PARP, and cleaved PARP antibodies were obtained from Biosystems Dogs (Shenyang, China). Other antibodies were all purchased from Beyotime Biotechnology (Shanghai, China).
10
Isolation and Characterization of Flavaspidic Acid BB
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Flavaspidic acid BB (BB, purity > 95%) was isolated from D. fragrans using silica gel column chromatography, Sephadex LH-20 column chromatography, and pre-HPLC with some modifications in the preliminary experiments [26 (link)–28 (link)]. Erythromycin (E, N0825A, 85%), mupirocin (MUP, 17,090,377, 95%), fusidic acid (FD, Z19J6Q1, 98%), cefazolin (190,501, 98%) and dimethyl sulfoxide (DMSO) were purchased from Dalian Meilun Biotechnology Inc. (Dalian, China), Sino-US Tianjin SK Pharmaceutical Inc. (Tianjin, China), Shanghai Yuanye Biotechnology Inc. (Shanghai, China), Guangdong South China Pharmaceutical Group Inc. (Guangzhou, China), and Sigma-Aldrich (Saint Louis, USA), respectively. Flavaspidic acid BB was dissolved in DMSO at a concentration of 51.2 mg/mL. E, MUP, and FD were dissolved in DMSO as a drug reserve solution (512 mg/ml) and stored at -20 °C.
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