The human chondrosarcoma cell line SW1353 cells used in our study were purchased from ATCC. The cells were routinely maintained in Leibovitz’s L-15 medium supplemented with 10% FBS and 1% compound antibiotics (100 units/mL penicillin, 100 μg/mL streptomycin). All cell cultures were kept in a humidified incubator environment with 95% air and 5% CO2 at 37°C. The AGE cell treatment reagent was freshly prepared in 150 mg/mL stock solution. Loratadine was obtained from Sigma-Aldrich, USA. For the treatment experiments, SW1353 cells were plated on different size plates and allowed to grow to the desired degree of confluence. The confluent cells were then treated with 50, 100, and 150 μg/mL AGEs for 24 hours. To test the effect of Loratadine, 25 and 50 µM concentrations of the drug were added to the cell media during the same time window as the AGEs. SW1353 cells were purchased from the American Type Culture Collection (Manassas, USA). Experiments were approved by the ethics committee of Jilin University.
Sw1353 cells
Manufactured by American Type Culture Collection
Sourced in United States
SW1353 cells are a human chondrosarcoma cell line derived from a grade II chondrosarcoma of the knee. These cells are available as a research tool for studying chondrosarcoma, a type of bone cancer that develops from cartilage cells.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Loratadine, by Merck Group (2 mentions)
Anti bax, by Cell Signaling Technology (2 mentions)
Anti p stat3, by Cell Signaling Technology (2 mentions)
Anti stat3, by Cell Signaling Technology (2 mentions)
L 15 medium, by Thermo Fisher Scientific (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Collagenase type 2, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Anti slug, by Abcam (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
Anti n cadherin, by Abcam (1 mentions)
Fetal bovine serum (fbs), by ScienCell (1 mentions)
14 protocols using sw1353 cells
1
Chondrosarcoma Cell Line Treatment with AGEs and Loratadine
2
Isolation of Osteoarthritic Chondrocytes
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Human chondrocytes were derived from 20 patients who underwent joint replacements for OA. These patients had no history of any other underlying diseases. The intercondylar cartilage consists of a non-weight-bearing area, as control cartilage, and a weight-bearing area, which is considered osteoarthritic cartilage. Cartilage cells from all patients were taken from the same tissue site and used separately. The position of the articular cartilage and patient information are shown in Supplementary Figures 1A,B . The cartilage tissue was shredded using disinfected instruments and incubated with type 2 collagenase (Sigma, CA, United States) for 6 h in a 37°C cell incubator. The residue was removed by filtration, followed by centrifugation at 1,000 rpm for 5 min to remove the supernatant. The cell pellet was resuspended and washed three times with phosphate-buffered saline (PBS). Cells were then transferred into a 10-cm petri dish and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, United States). All cells were used within three generations. HEK-293T cells [American Type Culture Collection (ATCC): CRL-1573), and SW1353 cells were obtained from the ATCC (Manassas, VA, United States) and cultured in DMEM with 10% FBS. All cells were grown in a 37°C incubator with 5% CO2.
3
Chondrocyte Cell Lines for Osteoarthritis
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The human articular chondrocyte cell line HC-a (Sciencell, Carlsbad, CA, USA) was maintained in DMEM supplemented with 15 % fetal bovine serum, plus antibiotics. SW1353 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and were maintained in L-15 medium (Gibco, Grand Island, NY). OUMS-27 cells, HCS-2/8 cells and JJ012 cells were kindly gifted from Dr. J Block (Rush Medical College, Chicago, IL, USA) and were cultured in Dulbecco Modified Eagle Medium (Hyclone, Logan, UT) supplemented with 10 % fetal calf serum (Gibco, Grand Island, NY) at 37 °C in a humidified atmosphere with 5 % CO2.
ATO was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The following antibodies were used in the experiments: anti-p-STAT3, anti-STAT3, anti-E-cadherin, anti-Vimentin, anti-Bax and anti-GAPDH were from Cell Signaling Technology (Beverly, MA, USA). anti-N-cadherin, anti-Slug, anti-MMP9 were from Abcam (USA). STAT3 siRNA were purchased from Suzhou GenePharma (Suzhou, China). Lipofectamin 3000 was bought from Origene (Rockville, MD, USA).
ATO was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The following antibodies were used in the experiments: anti-p-STAT3, anti-STAT3, anti-E-cadherin, anti-Vimentin, anti-Bax and anti-GAPDH were from Cell Signaling Technology (Beverly, MA, USA). anti-N-cadherin, anti-Slug, anti-MMP9 were from Abcam (USA). STAT3 siRNA were purchased from Suzhou GenePharma (Suzhou, China). Lipofectamin 3000 was bought from Origene (Rockville, MD, USA).
4
Cell Culture Conditions for Cancer Research
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The chondrosarcoma SW1353 cells, osteosarcoma Saos-2 cells and 293 cells were purchased from American Type Culture Collection (Manassas, VA, USA). Human XJH B lymphocytes were purchased from the Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). All cells were maintained at 37°C with 5% CO2 in an incubator with a constant humidity. PQQ was purchased from Sigma-Aldrich (Merck KGaA).
5
Chondrosarcoma Cell Treatment with IL-1β
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SW1353 cells (human chondrosarcoma cell line) purchased from the American type culture collection (Manassas, VA, USA) were cultured and treated with IL-1β according to previously described procedures [12] (link). In brief, the cells were maintained in DMEM with 10% FBS, glutamine, and penicillin/streptomycin. To induce MMP-13, IL-1β (10 ng/mL) with/without test compounds was added to the cells in serum-free DMEM for 24 h. MMP-13 released in the media was examined by Western blotting analysis using anti-MMP-13 antibody. All test compounds were initially dissolved in dimethyl sulfoxide (DMSO) and diluted with serum-free DMEM to adjust the final DMSO concentration to 0.1% (v/v). Cell viability was checked using MTT bioassay [13] (link). No effect on cell viability or the MMP-13 expression level was observed by the treatment of 0.1% DMSO.
6
Cytotoxicity Assay of EESS in SW1353 Chondrocytes
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SW1353 cells were obtained from the American Type Culture Collection (Manassas, VA, USA), and were cultured in Dulbecco’s modified Eagle’s medium (DMEM, WelGENE Inc., Daegu, Korea) containing 10% fetal bovine serum (FBS, WelGENE Inc.) and 100 U/mL penicillin and streptomycin (WelGENE Inc.) at 37 °C in humidified air with 5% CO2. The cytotoxicity of EESS to SW1353 chondrocytes was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In brief, SW1353 cells were treated with EESS or H2O2 (1 mM, Sigma-Aldrich Chemical Co.) alone, or pretreated with different concentrations of EESS for 1 h before H2O2 treatment. Afterward, the medium was removed, and 0.5 mg/mL of MTT (Sigma-Aldrich Chemical Co.) was added to each well, and incubated at 37 °C for 3 h. The supernatant was then replaced with an equal volume of DMSO, to dissolve blue formazan crystals for 10 min. The optical density was measured at a wavelength of 540 nm by microplate reader (Dynatech Laboratories, Chantilly, VA, USA). All experiments were performed in triplicate.
7
Human Chondrosarcoma Cell Culture
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Human chondrosarcoma cells (SW1353 cells) were obtained from American Type Culture Collection (ATCC; No. HTB-94, Manassas, VA, USA). The cells were grown in Leibovitz’s L-15 medium (Gibco, Grand Island, NY, USA) supplemented with 1% l-glutamine (Sigma-Aldrich, St. Louis, MO, USA), 1% antibiotic penicillin/streptomycin solution (Sigma-Aldrich, St. Louis, MO, USA), and 10% fetal bovine serum (HyClone, Logan, UT, USA). The cells were maintained at 37 °C in a humidified atmosphere without CO2, and the media was replaced every 3 days [36 (link)].
8
Culturing Chondrosarcoma Cells for Assays
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SW1353 cells (derived from human chondrosarcoma) were obtained from the American Type Culture Collection. The cells were cultured at 37°C in a humidified incubator in complete Leibovitz's L-15 medium (containing 10% fetal bovine serum to provide extra nutrition and penicillin and streptomycin to prevent bacterial contamination). For the MTS assay, cells were seeded in a 96-well plate. For PCR and Western blot analyses, cells were seeded in 6-well plates. Leibovitz's L-15 medium was used to support cell growth in an environment without CO2; thus, the culture bottle and plate were sealed as tightly as possible to minimize gas exchange with outside air.
9
Culturing Articular Chondrosarcoma Cells
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SW1353 cells, a human articular chondrosarcoma cell line, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) in the presence of penicillin (100 units/mL), streptomycin (100 μg/mL) and HEPES (25 mM) at 37°C in a humidified, 5% CO2/95% air, water-jacketed incubator.
10
Chondrogenic Differentiation of BMSCs
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All cells were cultured at 37°C in a humidified atmosphere with 5% CO2. SW1353 cells were obtained from the American Type Culture Collection, and TC28a2 cells were purchased from Sigma-Aldrich. SW1353 cells, TC28a2 cells, and human primary cells were cultured in DMEM with 10% CS supplemented with 1% PSG. BMSCs were purchased from Lonza and were cultured in human Mesenchymal Stem Cell (hMSC) Growth BulletKit Medium (MSCGM) (Lonza). In pellet culture of BMSCs, hMSC Chondrogenic Differentiation BulletKit Medium (Lonza) was used. Human IL-1β (PeproTech) and TGF-β3 (PeproTech) were used to treat cells. The compounds used in screenings and validation experiments were provided by Calibr (a Division of Scripps Research Institute). Mocetinostat for other experiments was obtained from LC Laboratories, and SR-18292 was purchased from Selleck Chemicals. Stocks of the compounds were diluted in DMSO. Cell viability was measured with Countess II FL Automated Cell Counter (Thermo Fisher Scientific) using Trypan Blue stain 0.4% (Thermo Fisher Scientific).
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