All cells were maintained in 10 cm dishes (Sarstedt) at 37°C and 5% CO2. HEK293A, HEK293T-GFP and 3T3 cells were grown in DMEM (Sarstedt) supplemented with 10% FBS (Sarstedt), 1% penicillin/streptomycin + l -glutamine (Gibco), 1% sodium pyruvate (Gibco) and 50 μM β-mercaptoethanol (Gibco). CH12-F3 mouse erythroleukemia B cells were grown in RPMI 1640 (Sarstedt) supplemented with 10% FBS (Sarstedt), 1% penicillin/streptomycin + l -glutamine (Gibco), 1% sodium pyruvate (Gibco), 5% NCTC-109 (Gibco) and 50 μM β-mercaptoethanol (Gibco).
Fetal bovine serum (fbs)
Manufactured by Sarstedt
Sourced in United States
Fetal Bovine Serum (FBS) is a cell culture supplement derived from the blood of bovine fetuses. It provides a wide range of essential nutrients, growth factors, and other components that support the growth and proliferation of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Nctc 109, by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin l glutamine, by Thermo Fisher Scientific (1 mentions)
10 cm dishes, by Sarstedt (1 mentions)
Dulbecco modified eagle medium (dmem), by Sarstedt (1 mentions)
Penicillin streptomycin, by GE Healthcare (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
L glutamine, by GE Healthcare (1 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
T75 flask, by Sarstedt (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Cd11b microbead isolation kit, by Miltenyi Biotec (1 mentions)
Penicillin streptomycin, by Sarstedt (1 mentions)
Dmem glutamax, by Sarstedt (1 mentions)
4 protocols using fetal bovine serum (fbs)
1
Cell Culture Conditions for Diverse Cell Lines
2
Culturing Human Neuroblastoma and Drosophila S2 Cells
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Human neuroblastoma SH-SY5Y cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; 4.5 g/L Glucose with L-Glutamine) supplemented with 1% (v/v) Penicillin/Streptomycin (GE Healthcare) and 10% Fetal Bovine Serum (FBS, Sigma) at 37°C, 5% CO2. Semi-adherent D. melanogaster S2 cells were maintained in Schneider’s medium containing L-Glutamine (Sigma) supplemented with 1% (v/v) Penicillin/Streptomycin/amphotericin B (GE Healthcare), 10% FBS, and maintained at 26°C in non-vented, adherent flasks (Sarstedt).
3
Isolation of Postnatal Mouse Microglia
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Mice aged P0–P3 were euthanized by decapitation, and brains were dissected into PBS on ice. Brains of 6–8 mice were pooled, centrifuged at 500 × g for 10 min at 4 °C and resuspended in 0.25% Trypsin-EDTA (Thermo Fisher Scientific) at 37 °C for 10 min. DNase I (Thermo Fisher Scientific) was added at 1 mg/ml to the solution, and the brains were digested for 10 more minutes at 37 °C. Trypsin was neutralized by adding DMEM + GlutaMAX (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Thermo Fisher Scientific), and cells were passed through a 70 µm cell strainer. Cells were centrifuged at 500 × g for 10 min at 4 C, resuspended in DMEM + GlutaMAX with 10% FBS 1% penicillin/streptomycin and cultured in T-75 flasks (Sarstedt), pre-coated with 2 µg/ml Poly-L Lysine (PLL, Provitro) at 37 °C in a humidified incubator with 5% CO2 for 5–7 days until confluence was reached. Mixed glial cells were shaken for 30 min at 180 rpm, the supernatant was collected and the medium was changed, and then cells were shaken for at least 2 h at 220 rpm and the supernatant was collected and the medium was changed again. CD11b+ microglia were isolated from the collected supernatant using the CD11b Microbead Isolation kit (Miltenyi) according to the manufacturer’s instruction and seeded onto PLL-coated plates.
4
Immunomodulatory Effects of hPDLSCs on T Cells
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Primary hPDLSCs were seeded in 6-well plates at a density of 2.5 × 105 cells per well in 3 ml DMEM (Sigma-Aldrich, St. Louis, USA) containing 10% FBS (Gibco, Carlsbad, USA) and 1% P/S (Gibco, Carlsbad, USA). After 24 h, hPDLSCs were stimulated as indicated above. After 48 h, medium was changed to RPMI-1640 (Sigma-Aldrich, St. Louis, USA) containing 10% FBS and 1% P/S. Transwell (TC) inserts (0.4 µm pore size, Sarstedt, Nürnbrecht, Germany) containing 1 × 106 allogenic CD4+ T cells were inserted into each well. CD4+ T lymphocyte proliferation was induced by 10 µg/ml phytohemagglutinin-L (PHA-L, eBioscience, San Diego, USA) in the presence or the absence of different cytokines. After five days, CD4+ T lymphocytes proliferation and apoptosis were analyzed by flow cytometry. CD4+ T lymphocytes cultured in the absence of hPDLSCs served as reference.
Additionally, hPDLSCs in co-culture were treated with pharmacological inhibitors against IDO-1, PD-L1, and PTGS-2, as mentioned above. We aimed to clarify the role of these immunomediators on hPDLSCs’ caused effects on CD4+ T lymphocyte proliferation and apoptosis at different conditions. After 5 days incubation, CD4+ T lymphocyte proliferation and apoptosis were assessed by flow cytometry analysis.
Additionally, hPDLSCs in co-culture were treated with pharmacological inhibitors against IDO-1, PD-L1, and PTGS-2, as mentioned above. We aimed to clarify the role of these immunomediators on hPDLSCs’ caused effects on CD4+ T lymphocyte proliferation and apoptosis at different conditions. After 5 days incubation, CD4+ T lymphocyte proliferation and apoptosis were assessed by flow cytometry analysis.
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