HL60 cells were cultured in RPMI-1640 medium (LifeTechnologies) supplemented with 10% of heat-inactivated Fetal Calf Serum (FCS) (lot n°S11060S181, Dominique Dutscher) containing Penicillin-Streptomycin (50 UI/ml and 50 µg/ml) (GIBCO®). HL60 cells were seeded every 2–3 days at 100,000 cells/mL in 5 mL in 25 cm2 flasks (BD Falcon). H1975 cells were cultured in RPMI-1640 medium supplemented with 10% Fetal Calf Serum (FCS) (lot n°S11060S181, Dominique Dutscher) and 1% Sodium Pyruvate and Hepes (Lifetechnologies). H1975 cells were seeded twice a week at 300,000 cells/mL in 20 mL in a 75 cm2 flask (BD Falcon). All cell lines were incubated at 37 °C and 5% CO2.
Fetal calf serum (fcs)
Manufactured by Dutscher
Sourced in France, United States
Fetal calf serum is a cell culture supplement derived from the blood of unborn calves. It contains a complex mixture of proteins, growth factors, and other components that support the growth and proliferation of a wide variety of cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Dutscher (3 mentions)
Meso quickplex sq 120, by Mesoscale (2 mentions)
V plex system, by Mesoscale (2 mentions)
Methylcellulose, by Bio-Techne (2 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (2 mentions)
Mda mb 231, by American Type Culture Collection (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
Hepes, by Dutscher (2 mentions)
Trypan blue, by Merck Group (2 mentions)
Collagenase crude type 1 a, by Merck Group (2 mentions)
Fluorescein isothiocyanate fitc dextran, by Merck Group (2 mentions)
Ile pro ile, by Merck Group (1 mentions)
Rpmi 1640 medium, by Dutscher (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Hpi t, by Philips (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by R&D Systems (1 mentions)
33 protocols using fetal calf serum (fcs)
1
Cell Culture Protocols for HL60 and H1975 Cell Lines
2
Genotoxicity Assay with V79 and SHE Cells
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V79 cells (lung fibroblast from Chinese hamster, ATCC, USA, reference CCL-93) were selected for this study as they are one of the cell models recommended in OCDE guideline number 487 for use in the in vitro micronucleus assay. Cells were grown in Dulbecco's MEM (DMEM; Invitrogen, France), supplemented with 10% fetal calf serum (Dutscher, France) and 0.5% Penicillin/Streptomycin (5000 U-5000 μg/mL, Invitrogen, France). Cells were incubated at 37°C with 10% CO2, as recommended by the supplier for optimal culture with our medium.
Syrian hamster embryo (SHE) cell cultures were used as they are normal diploid cells, nongenetically modified, metabolically competent, and p53 effective and there is no known difference with those constituting the organism where they come from. They have been demonstrated to be suitable for genotoxicity assays [37 (link), 38 (link)]. Cells were established from individual 13-day gestation foetuses (inbred colony, INRS, France). The culture medium used was Dulbecco's MEM (DMEM; Invitrogen, France), supplemented with 17% fetal calf serum (Dutscher, France) and 0.5% Penicillin/Streptomycin (5000 U-5000 μg/mL, Invitrogen, France). Cells were incubated at 37°C and 10% CO2.
Syrian hamster embryo (SHE) cell cultures were used as they are normal diploid cells, nongenetically modified, metabolically competent, and p53 effective and there is no known difference with those constituting the organism where they come from. They have been demonstrated to be suitable for genotoxicity assays [37 (link), 38 (link)]. Cells were established from individual 13-day gestation foetuses (inbred colony, INRS, France). The culture medium used was Dulbecco's MEM (DMEM; Invitrogen, France), supplemented with 17% fetal calf serum (Dutscher, France) and 0.5% Penicillin/Streptomycin (5000 U-5000 μg/mL, Invitrogen, France). Cells were incubated at 37°C and 10% CO2.
3
Murine Cancer Models: Subcutaneous Implantation
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CT26 American Type Culture Collection (ATCC) murine colon cancer cells (USA) were cultured in RPMI 1640 (Dutscher, France) + 10% fetal calf serum (Dutscher, France) (37 °C, 5% carbon dioxide and 95% humidity). B16-F10 murine melanoma cancer cells (USA) were cultured in DMEM (Dutscher, France) + L-Glutamine + red phenol + glucose (4.5 g/l) + 10% fetal calf serum (Dutscher, France) (37 °C, 5% carbon dioxide and 95% humidity).
The day before mice were injected with cancer cells. These cells were contacted with trypsin and diluted to ½. The unit injection included 5 × 105 CT26 cells in 100 μl of NaCl, or 1 × 106 B16-F10 cells 100 μl of NaCl, in performed subcutaneously on the right flank of immunocompetent BALB/c female and C57BL female mice and 8-week immunosuppressed athymic BALB/c nude mice (Charles River Laboratories, Saint-Germain-des-Monts, France). During the entire duration of the experiment, mice were housed in our approved animal facility (Centre Georges-François Leclerc, Dijon, FRANCE). The mice were sacrificed by cervical dislocation after Isoflurane 2.5% anesthesia as soon as a limit point was reached (Tumoral Volume (TV) ≥1500 mm3, pain, significant necrosis).
Before experimentation, the small animal ethics committee and the Ministry of Higher Education and Research validated the project.
The day before mice were injected with cancer cells. These cells were contacted with trypsin and diluted to ½. The unit injection included 5 × 105 CT26 cells in 100 μl of NaCl, or 1 × 106 B16-F10 cells 100 μl of NaCl, in performed subcutaneously on the right flank of immunocompetent BALB/c female and C57BL female mice and 8-week immunosuppressed athymic BALB/c nude mice (Charles River Laboratories, Saint-Germain-des-Monts, France). During the entire duration of the experiment, mice were housed in our approved animal facility (Centre Georges-François Leclerc, Dijon, FRANCE). The mice were sacrificed by cervical dislocation after Isoflurane 2.5% anesthesia as soon as a limit point was reached (Tumoral Volume (TV) ≥1500 mm3, pain, significant necrosis).
Before experimentation, the small animal ethics committee and the Ministry of Higher Education and Research validated the project.
4
Measuring P. distasonis-induced GLP-1 secretion
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The ability of the P. distasonis strains to induce the secretion of GLP-1 was measured using the neuroendocrine murine cell line STC-1 (ATCC CRL-3254™), as previously described [14 (link),35 (link)] Cells were grown at 37 °C under 5% CO2 in DMEM (Life Technologies, Saint Aubin, France), supplemented with fetal calf serum (10%, Dutscher, Brumath, France), L-glutamine (5 mM) and streptomycin and penicillin (100 µg/mL). For bacterial stimulation, cells were grown in 12-well plates (200,000 cells/well) for 72 h, washed twice with PBS and resuspended in 400 µL of 20 mM Hepes/20 mM Tris pH 7.4 buffer containing 140 mM NaCl, 4.5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 10 mM glucose. Cells were stimulated (or not) with the bacteria (10:1 bacteria/cell) or with butyrate (10 mM final) as a positive control at 37 °C under 5% CO2. After 8 h stimulation, the supernatants were collected and centrifuged (10 min at 8000× g). DPP-IV enzyme inhibitor (Ile-Pro-Ile, Sigma-Aldrich Germany) was added (100 µM), and samples were stored at −20 °C. The level of active GLP-1 was measured using the V-Plex system and MESO QuickPlex SQ 120 (MesoScale Diagnostics, Rockville, MD, USA).
5
Characterization of STS Cell Lines
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All of the STS cell lines used in this study were derived from surgical specimens of patients with STS who underwent surgery at Institut Bergonié, Bordeaux, France and who gave written informed consent (Table 1 ) as previously described24 (link). Each cell line was characterized by array comparative genomic hybridization for every 10 replicates to verify that its genomic profile was still representative of the originating tumor sample. Cells were grown in RPMI medium 1640 (Sigma Life Technologies, Saint Louis, MO) in the presence of 10% fetal calf serum (Dutscher, France) in flasks. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2.
6
Coral Propagation and Fibroblast Culture
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Nubbins of
Stylophora pistillata were collected from five mother colonies maintained in the aquaria facilities of the Centre Scientifique de Monaco. Two small nubbins (3–5 cm long) were cut off from each mother colony, and were allowed to heal for four weeks in 15 L open system tanks before the experiments. Corals were maintained in the same conditions as the mother colonies,
i.e. at 25°C, under a photosynthetic active radiation of 200 µmol photon.m
-2.s
-1 provided by 400 W metal halide lamps (HPIT, Philips) and were fed twice a week with
Artemia salina nauplii. Seawater in the tanks was continuously renewed at a rate of 10 L.h
-1.
Immortalized skin fibroblasts (BJ-EHLT cells) were kindly provided by E. Gilson’s lab (IRCAN) and cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Villebon-sur-Yvette, France) supplemented with 10% heat-inactivated fetal calf serum (Dutscher, Brumath, France) at 37°C in an atmosphere of 5% CO
2, as previously described (
Biroccio
et al., 2013 ).
Stylophora pistillata were collected from five mother colonies maintained in the aquaria facilities of the Centre Scientifique de Monaco. Two small nubbins (3–5 cm long) were cut off from each mother colony, and were allowed to heal for four weeks in 15 L open system tanks before the experiments. Corals were maintained in the same conditions as the mother colonies,
i.e. at 25°C, under a photosynthetic active radiation of 200 µmol photon.m
-2.s
-1 provided by 400 W metal halide lamps (HPIT, Philips) and were fed twice a week with
Artemia salina nauplii. Seawater in the tanks was continuously renewed at a rate of 10 L.h
-1.
Immortalized skin fibroblasts (BJ-EHLT cells) were kindly provided by E. Gilson’s lab (IRCAN) and cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Villebon-sur-Yvette, France) supplemented with 10% heat-inactivated fetal calf serum (Dutscher, Brumath, France) at 37°C in an atmosphere of 5% CO
2, as previously described (
et al., 2013
7
Chondrosarcoma Spheroid Formation Protocol
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Human HEMC-SS chondrosarcoma cell line was obtained from the European Collection of Authenticated Cell Cultures and cultured in DMEM/F12 medium (Life Technologies) supplemented with 10% fetal calf serum (Dutscher) and 4 μg/mL gentamicin.
To generate HEMC-SS spheroids, cells were harvested from culture flasks by trypsinization and seeded in 96-well non-adherent plates (Nunc) with 100 μL of 0.5% methylcellulose (Bio-Techne) diluted in the corresponding medium at a density of 1000 cells per well. Cultures were maintained in a 5% CO2 humidified environment at 37°C.
Spheroid cell numbers were determined at different times (Day 1 to Day 10) after cell dissociation by incubation with Trypsin-EDTA (Life Technologies) for 5 minutes at 37°C.
To generate HEMC-SS spheroids, cells were harvested from culture flasks by trypsinization and seeded in 96-well non-adherent plates (Nunc) with 100 μL of 0.5% methylcellulose (Bio-Techne) diluted in the corresponding medium at a density of 1000 cells per well. Cultures were maintained in a 5% CO2 humidified environment at 37°C.
Spheroid cell numbers were determined at different times (Day 1 to Day 10) after cell dissociation by incubation with Trypsin-EDTA (Life Technologies) for 5 minutes at 37°C.
8
Establishment and Maintenance of DDLPS and Leiomyosarcoma Cell Lines
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The DDLPS (IB115 and IB111) and leiomyosarcoma (IB136 and IB112) cell lines used in this study were derived from human surgical specimens (Institut Bergonié, Bordeaux, France) after obtaining patient consent. The MDM2 antagonist-resistant IB115 [P4] cell line was derived from IB115 cells through repeated exposure to the nutlin compound RG7388, as previously described [12 (link)]. Cells were maintained in RPMI medium 1640 (Sigma Life Technologies, St. Louis, MO, USA) with 10% fetal calf serum (Dutscher, France) in a humidified incubator containing 5% CO2 maintained at 37 °C.
9
Generating HEMC-SS Spheroids for Research
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Human HEMC-SS CHS cell line was obtained from the European Collection of Authenticated Cell Cultures, and cultured in DMEM/F12 medium (Life Technologies, Carlsab, CA, USA) supplemented with 10% fetal calf serum (Dutscher) and 4 μg/mL gentamicin.
To generate HEMC-SS spheroids, cells were harvested from culture flasks by trypsinization, and seeded in 96-well non-adherent plates (Nunc) with 100 μL of 0.5% methylcellulose (Bio-Techne) diluted in the whole culture medium at a density of 1000 cells per well. Cultures were maintained in a 5% CO2 humidified environment at 37°C.
Spheroid doubling time was determined from exponential linear regression of cell counts from spheroids dissociated by incubation with trypsin-EDTA (Life Technologies) for 5 minutes at 37°C
To generate HEMC-SS spheroids, cells were harvested from culture flasks by trypsinization, and seeded in 96-well non-adherent plates (Nunc) with 100 μL of 0.5% methylcellulose (Bio-Techne) diluted in the whole culture medium at a density of 1000 cells per well. Cultures were maintained in a 5% CO2 humidified environment at 37°C.
Spheroid doubling time was determined from exponential linear regression of cell counts from spheroids dissociated by incubation with trypsin-EDTA (Life Technologies) for 5 minutes at 37°C
10
Saos-2 cell culture impacts S. aureus biofilm
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The Saos-2 cell line (ATCC®HTB-85TM) was cultured at 37°C in a 5% CO2 humidified atmosphere in Dulbecco’s modified Eagle’s medium (DMEM-Gibco) supplemented with 10% fetal calf serum (Dutscher) and 1% antibiotic solution PenStrep®(Gibco) considered as standard medium (SM). Saos-2 cells were grown to 60–80% confluence in SM then rinsed with sterile Dulbecco’s Phosphate Buffered Saline (DPBS, Gibco) to eliminate antibiotics. Saos-2 cells were further incubated with DMEM and 10% fetal calf serum, without antibiotics, supplemented or not with recombinant human Tumor Necrosis Factor α (TNF α) at 20 ng/mL (R&D Systems) in 25 cm2 flasks. After 72 h of incubation, collected supernatants of Saos-2 cells were applied to S. aureus cultures in minimal media (50% of supernatants and 50% of MM, named SN 50). In each condition, the initial quantity of bacteria was ∼106 CFU/mL. Bacterial adhesion was evaluated after 24 h of contact in the static biofilm models (described above).
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