Human chondrocyte cells CHON002, derived from a female patient (American Type Culture Collection, Manassas, VA, USA), were cultured in 100 mm dishes containing 10 ml of Dulbecco's modified Eagle's medium (DMEM; American Type Culture Collection, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS; Premium Fetal Bovine Serum, Lonza, Basel, Switzerland) and 0.1 mg/ml G-418 sulfate (cell culture tested, Calbiochem, San Diego, USA). Cultures were incubated at 37℃ in humidified air with 5% CO2. When the chondrocyte cultures reached 80% confluence, the cells were washed with phosphate-buffered saline (PBS; Dulbecco's Phosphate-Buffered Saline, Invitrogen, Carlsbad, USA), treated with a Trypsin-Versene (EDTA) mixture (Lonza), and subcultured. The medium was routinely changed every 2 days. At passage 6, chondrocytes were cultured at 37℃ for 24 h in DMEM containing 1% FBS and 0.1 mg/ml G-418 sulfate, and stabilized. After a 24 h culture, chondrocytes were washed three times in PBS, and collected by centrifugation (1,000×g, 3 min) after each wash.
Trypsin versene edta mixture
Manufactured by Lonza
Sourced in United States, Germany
Trypsin-Versene (EDTA) mixture is a laboratory reagent that contains a combination of trypsin and EDTA (ethylenediaminetetraacetic acid). Trypsin is a proteolytic enzyme that helps in the dissociation and detachment of adherent cells from cell culture surfaces, while EDTA is a chelating agent that aids in the disruption of cell-cell and cell-substrate interactions. This mixture is commonly used in cell culture procedures to facilitate the harvesting, passaging, and sub-culturing of adherent cells.
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Lab products found in correlation
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (1 mentions)
Chon002, by American Type Culture Collection (1 mentions)
Alexa 647 dye, by Thermo Fisher Scientific (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
35 mm cell imaging dishes, by Eppendorf (1 mentions)
Dmi3000b inverted microscope, by Leica (1 mentions)
Imark plate reader, by Bio-Rad (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
Nhdf cells, by PromoCell (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Lonza (1 mentions)
Dmem f12 medium, by Lonza (1 mentions)
Penicillin streptomycin amphotericin b mixture, by Lonza (1 mentions)
4 protocols using trypsin versene edta mixture
1
Chondrocyte Cell Culture Protocol
2
Macrophage Ovalbumin Internalization Assay
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Chicken ovalbumin was fluorescently labeled with the Alexa647 dye using a bioconjugation kit (ThermoFisher). Bone-marrow derived macrophages were prepared as described in the previous paragraph. 2.104 cells were harvested, and plated on plastic (96-flat-well plate), let to adhere for 2 hr, then exposed to MHV (or not) for 24 hr. Macrophages were then infected with varying concentrations of fluorescently-labeled chicken ovalbumin for 2 hr, harvested with a 15 min exposure to a solution of trypsin-versene-EDTA mixture (Lonza), washed with FACS buffer (PBS/4%FBS/0.1%Sodium Azide) and immediately analyzed by flow cytometry.
3
Evaluation of Cell Lines' Cytotoxicity
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Human osteosarcoma (HOS) and MCF7 cell lines were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany) and Normal Human Dermal Fibroblasts (NHDF) cell line from PromoCell, (Heidelberg, Germany); Eagle’s Minimal Essential Medium alpha (aMEM), Dulbecco’s Modified Eagle Medium (DMEM) without phenol red, 1% Penicillin–Streptomycin–Amphotericin B mixture (10K/10K/25 µg in 100 mL) and Trypsin–Versene (EDTA) mixture from Lonza, Verviers, Begium; fetal bovine serum (FBS) from Biochrom GmbH, Germany, CellTiter 96® Aqueous One Solution Cell Proliferation Assay from Promega (Madison, WI, USA); Tryple from Gibco, (Langley, VA, USA); LIVE/DEAD Viability/Cytotoxicity Kit and phosphate buffered saline (PBS) from Invitrogen, (Eugene, OR, USA), 35-mm Cell Imaging Dishes from Eppendorf (Hamburg, Germany).
Absorbance at 490 nm from MTS assay was measured with an iMark plate reader (Biorad, Hercules, CA, USA) and images for live/dead staining and wound scratch assays were recorded with a Leica DMI 3000B inverted microscope (Wetzlar, Germany).
Absorbance at 490 nm from MTS assay was measured with an iMark plate reader (Biorad, Hercules, CA, USA) and images for live/dead staining and wound scratch assays were recorded with a Leica DMI 3000B inverted microscope (Wetzlar, Germany).
4
Cytotoxicity Assay with NHDF Cells
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For cytotoxicity tests, normal human dermal fibroblasts (NHDF) cells (from PromoCell) at a passage number lower than 10 were grown in T25 culture flasks with 10 mL of DMEM:F12 medium (from Lonza) supplemented with 10% fetal bovine serum (FBS, Gibco), 1 mM sodium pyruvate (Lonza) and 1 % Penicillin-Streptomycin-Amphotericin B mixture (10K/10K/25 µg in 100 mL, Lonza). Cell growing was done at 37 °C and 5 % CO 2 under humidified atmosphere and medium was changed with fresh one every 4 days. NHDF cells were harvested by trypsinization with Trypsin-Versene (EDTA) mixture (Lonza), washed with phosphate buffered saline (PBS, Invitrogen) centrifuged at 200 rpm for 5 min, counted and seeded at a density of 5x10 3 cells/well in 96 well plates with 100 µL/well of the above specified medium. Cells were incubated and were allowed to adhere in humidified atmosphere at 37°C until the next day.
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