Immediately after treatment, osteoblast-like cells (osteosarcoma cells, MG-63; ATCC CRL-1427; LGC Standards, Wesel, Germany) were seeded on top of implants. Before seeding the cells, 1.3 mL of media was placed in each microplate well containing the implants. Then, 150 µL of cell suspension, adjusted to 1.5 × 105 cells/mL, was pipetted in a meandering pattern above prepared specimens. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) without phenol red and any antibiotics (to allow concomitant biofilm regrowth) at 37°C in a humidified atmosphere with 5% CO2 for 5 days, without media change. Cells were cultivated in tissue culture flasks (Eppendorf Italia Srl, Milan, Italy), were split at approximately 80% of confluence by a trypsin (0.05%)/EDTA (0.02%) solution (Sigma-Aldrich, Milan, Italy), and stopped with DMEM containing 20% FBS to attain an adequate number of cells.
Trypsin 0.05 edta 0 02 solution
Manufactured by Merck Group
Sourced in Italy
Trypsin (0.05%)/EDTA (0.02%) solution is a laboratory reagent used for the dissociation and detachment of adherent cells in cell culture applications. The solution contains trypsin, a proteolytic enzyme, and EDTA, a chelating agent, which work together to break down the extracellular matrix and cell-cell adhesions, allowing cells to be harvested and subcultured.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Mg 63, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Dutscher (1 mentions)
Penicillin streptomycin, by Dutscher (1 mentions)
Hepes buffer, by Thermo Fisher Scientific (1 mentions)
Rpmi glutamine medium, by Merck Group (1 mentions)
High glucose dmem medium, by Dutscher (1 mentions)
Zeocin, by InvivoGen (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Hepes, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
3 protocols using trypsin 0.05 edta 0 02 solution
1
Osteoblast-like Cell Seeding on Implants
2
HT-29 Epithelial Cell Immunomodulation
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HT-29 human epithelial cells were used for immunomodulation assays; either the parental lineage (HT-29, colon adenocarcinoma; ATCC HTB-38) or a lineage transfected with the secreted alkaline phosphatase (SEAP) reporter gene for NF-kB activation monitoring (HT-29/kb-seap-25) (Lakhdari et al., 2010) . The reporter HT-29/kb-seap-25 cells were cultured in RPMI-Glutamine medium (Sigma-Aldrich), supplemented with 10% fetal bovine serum (Corning), 1% non-essential amino acids, 1% sodium pyruvate, 1% HEPES buffer (Thermofisher Scientific), and 1% penicillin-streptomycin (Lonza) according to Lakhdari et al. (2010) . The parental HT-29 cells were cultured in high-glucose DMEM medium (Dominique Dutscher) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Do Carmo et al., 2017) . For subcultures, cells were rinsed with DPBS (Thermofisher Scientific) and detached with a trypsin (0.05%) -EDTA (0.02%) solution (Sigma). Periodically, 100 µg/mL Zeocin (Invivogen) was applied to the HT-29/kb-seap-25 cell culture in order to maintain selective pressure on the cells containing the transfected plasmid.
3
Murine Stromal Cell Line MS-5 Culture
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The murine stromal cell line MS-5 was obtained from DSMZ (German Collection of Microorganisms and Cell Cultures) Leibniz Institute (Braunschweig, Germany) and cultured in DMEM (Life Technologies, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS, PAA Laboratories, Pasching, Austria), 100 U/ml penicillin-100 µg/ml streptomycin (Sigma-Aldrich, Darmstadt, Germany), 2 mM L-glutamine (Sigma-Aldrich), 10 mM HEPES (Sigma-Aldrich), 1 mM MEM sodium pyruvate, 1% MEM vitamins solution and 50 µM 2-mercaptoethanol (Gibco Invitrogen, Darmstadt, Germany) at 37°C and 5% CO 2 . The cells were routinely subcultured by trypsinization using a trypsin (0.05%)-EDTA (0.02%) solution (Sigma-Aldrich).
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