Primary chondrocytes were harvested according to a previous protocol.24 The New Zealand rabbits were purchased from the Third Military Medical University (Chongqing, China). The experiment was approved by Animal Care and Use Committee of the Third Military Medical University. Articular cartilage was removed from the knee joint of rabbits (1 month old, n = 3) with a sterile blade and minced. Cartilage pieces were digested with 0.2% w/v type collagenase (Sigma, St. Louis, MO, USA) in DMEM/F12 (Gibco, Grand Island, NY, USA) supplemented with 1% v/v penicillin‐streptomycin (Gibco) at 37°C in a 5% CO2 incubator overnight. Digested tissue was filtered with a 40‐μm‐cell strainer (BD Bioscience, Franklin Lakes, NJ, USA) followed by centrifugation at 400 g for 5 minutes and resuspended in DMEM/F12 supplemented with 10% v/v foetal bovine serum (Tbdscience, Tianjin, China) and 1% v/v penicillin‐streptomycin. Medium was changed every 3 days. Cells were passaged at 80%‐90% confluence, and the passage 2 (P2) cells were used in the following experiments. For human TGF‐β1 stimulation, chondrocytes were treated with 5 ng/mL TGF‐β1 (Peprotech, Rocky Hill, NJ, USA) for 24 hours.
Type collagenase
Manufactured by Merck Group
Sourced in United States
Type collagenase is an enzyme that cleaves collagen, the primary structural protein in the extracellular matrix. It is commonly used in cell culture and tissue engineering applications to facilitate the dissociation and isolation of cells from collagen-rich tissues.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Foetal bovine serum (fbs), by TBD Science (1 mentions)
Dmem f 12, by TBD Science (1 mentions)
Penicillin streptomycin, by TBD Science (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
40 μm cell strainer, by BD (1 mentions)
Gw6471, by MedChemExpress (1 mentions)
3 protocols using type collagenase
1
Isolation of Rabbit Chondrocytes
2
Isolation and Culture of Mouse Primary Hepatocytes
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Mouse primary hepatocytes (MPHs) were isolated from livers of male C57BL/6J mice, PPARα−/− mice or Klf16alb−/− mice (8 weeks old) as previously reported. Briefly, liver cells were isolated by injection of 0.5 mg/mL type collagenase (Sigma) through the inferior vena cava after anaesthesia in mice. Trypan blue dyes were used to detect cell activity (70% or more). Then, MPHs were cultured in RPMI-1640 Medium containing 10% fetal bovine serun (FBS), 100 units/mL penicillin and 0.1 mg/mL streptomycin. MPHs were infected with Ad-Gfp, Ad-Klf16, Ad-shLuci, Ad-shKlf16 or Ad-Pparα for 24 hours, and the medium was replaced with oleic acid and palmitic acid (OA and PA) containing fresh medium for another 24 hours. To inhibit PPARα, MPHs were treated with GW6471 (10 µM, MedChemExpress). Cells were then collected for further analysis.
3
Isolation and Culture of Synovial Cells
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Isolation and Culture of FLS Normal synovium was obtained from meniscal knee arthroscopic surgery patients, and RA synovium was obtained from RA patients undergoing knee arthroplasty. Tissue samples were digested in type collagenase (Sigma) in DMEM-F12(Gibco) for 1-2 hours and then gently shaken at 37°C for 2 hours.
After centrifugation, the cells were cultured in a DMEM-F12(Gibco) medium containing 10% FBS, 100 U/ml penicillin, 100µg/ml streptomycin at 37°C, and 5% CO 2 . Cells of passages 3-6 were used in this study.
After centrifugation, the cells were cultured in a DMEM-F12(Gibco) medium containing 10% FBS, 100 U/ml penicillin, 100µg/ml streptomycin at 37°C, and 5% CO 2 . Cells of passages 3-6 were used in this study.
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