Fetal bovine serum (fbs)

Manufactured by LGC

Sourced in Brazil, United States

FBS is a cell culture supplement derived from the blood of fetal bovine. It provides various nutrients, growth factors, and other components that support the growth and proliferation of cultured cells.

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36 protocols using fetal bovine serum (fbs)

Cell Culture Protocols for Cancer Research

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All cell lines used (human squamous cell carcinoma A431, human pharynx carci-noma FaDU and green monkey kidney Vero CCL-81) were obtained from ATCC (Manas-sas, VA, USA) and grown at 37 °C with 90% humidity and 5% CO₂ incubator in DMEM High Glucose containing 1 mM sodium pyruvate from Gibco (Grand Island, NY, USA); they were also supplemented with 5% Fetal bovine serum from LGC Biotechnology (Cotia, SP, Brazil).
Trypsin solution from Gibco (New York, NY, USA). Hoechst 33,342 was obtained from Sigma Aldrich (New York, NY, USA). T25 Tissue culture flasks and multi-well plates were obtained from Kasvi (Curitiba, PR, Brazil) and TPP (Trasadingen, Switzerland). Resazurin viability kit was purchased from Sigma (St. Louis, MO, USA).

Malagrino T.R., Godoy A.P., Barbosa J.M., Lima A.G., Sousa N.C., Pedrotti J.J., Garcia P.S., Paniago R.M., Andrade L.M., Domingues S.H., Silva W.M., Ribeiro H, & Taha-Tijerina J. (2022). Multifunctional Hybrid MoS2-PEGylated/Au Nanostructures with Potential Theranostic Applications in Biomedicine. Nanomaterials, 12(12), 2053.

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SH-SY5Y Cell Culture Protocol

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Human SH-SY5Y cells (LGC Standards S.r.l., Sesto San Giovanni, Italy) were cultured as monolayers in polystyrene dishes in Dulbecco's modified Eagle's medium (DMEM) containing 15% heat-inactivated fetal bovine serum, 1% L-glutamine (200 mM), 1% sodium pyruvate (100 mM), 100 IU/ml penicillin, and 100 μg/ml streptomycin. All of the above reagents were purchased from Invitrogen (Milan, Italy). Cells were grown in a humidified incubator at 37°C in a 5% CO₂ atmosphere, and the medium was changed every 2 days. Each experiment was performed using cells (passages 15–30) plated on multiwell plates. After 24 h and 48 h of cell seeding, cells were incubated with A1254 (stock solution; 1 mg/ml) in DMEM without serum.

Cocco S., Secondo A., Del Viscovo A., Procaccini C., Formisano L., Franco C., Esposito A., Scorziello A., Matarese G., Di Renzo G, & Canzoniero L.M. (2015). Polychlorinated Biphenyls Induce Mitochondrial Dysfunction in SH-SY5Y Neuroblastoma Cells. PLoS ONE, 10(6), e0129481.

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Cultivation and Quantification of G8 Macrophages

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The G8 macrophage strain was maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (LGC Biotechnology), 100 U/mL penicillin, 100 mg/mL streptomycin (Sigma-Aldrich), and 20 mM HEPES (Sigma-Aldrich). Cultured cells were maintained at 37 °C in a humidified atmosphere containing 5% CO 2 for plating the J774. G8 cells, the medium used in the culture bottles was added a 50 mL conical tube. Adhered cells were trypsinized with trypsin-EDTA solution (1X) and placed in a conical tube with the medium removed previously.
Then, the tube was centrifuged at 400 g for 5 minutes, and the pellet was resuspended in 1 mL of RPMI-1640 medium. The number of cells was obtained by counting in a Neubauer chamber at a 1:100 dilution in 0.4% trypan blue solution (LGC Biotechnology). Next, cells Chart 1. Samples of plants from the Restinga de Jurubatiba sectored according to species, family, plant part, fraction and corresponding acronym. were plated in 96-well microplates at a concentration of 1x10 5 and incubated at 37 °C in a 5% CO 2 atmosphere until use.

Marques-Santos F., Amendoeira M.R.R., Galvão R.M.S., Rocha L.M, & Faria R.X. (2023). Comparative evaluation of plant extract effects on peritoneal, medullary and J774 cells. G8 macrophages. Brazilian journal of biology = Revista brasleira de biologia, 83.

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MDA-MB 231 Breast Cancer Cell Culture

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The human breast cancer cell line MDA-MB 231 was obtained from the American Type Culture Collection (ATCC HTB-26). Cells were routinely grown in Roswell Park Memorial Institute (RPMI) essential medium (Sigma Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (LGC Biotechnology, Cotia, SP, Brazil) and 1% penicillin–streptomycin (Sigma Aldrich, St. Louis, MO, USA) in a humidified 5% CO₂ atmosphere at 37 °C. Cells were cultured up to 70–100% confluence. The compounds were dissolved in dimethyl sulfoxide (DMSO) (Sigma Aldrich, St. Louis, MO, USA) in a 1 mM stock solution, and the concentrations used were dissolved in RPMI at different concentrations. The DMSO concentration did not exceed 0.1% in the final solution and had no effect per se.

Guerra F.S., Dias F.R., Cunha A.C, & Fernandes P.D. (2021). Benzo[f]indole-4,9-dione Derivatives Effectively Inhibit the Growth of Triple-Negative Breast Cancer. Molecules, 26(15), 4414.

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Culturing Leishmania infantum Promastigotes and Murine Macrophages

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Promastigotes of L. infantum strains MHOM/BR/1972/LD and MHOM/MA/67/ITMAP-263 were maintained at 27°C in Schneider’s medium (Sigma) supplemented with 100 mL of heat-inactivated fetal bovine serum (FBS), streptomycin, and penicillin (100 mg mL^-1; 100 μM mL^-1) (LGC Biotecnologia) and 10% of sterile masculine human urine, in 25 cm² culture bottles (TPP). Peritoneal macrophages were obtained as previously described [34 (link)]. Briefly, cells were collected from the peritoneal cavity of six to eight weeks old male Swiss mice, previously stimulated with thioglycolate 3%. For this, PBS (pH 7.4) was injected into the peritoneal cavity, followed by a slight massage, and the content was collected using a syringe (5 mL). These cells were cultivated in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 25 mM HEPES, 2 mM L-glutamine, 1% penicillin/streptomycin and incubated at 37°C in a 5% CO₂-air mixture in 96 well-plates or 24 well-plates.

Clementino L.D., Fernandes G.F., Prokopczyk I.M., Laurindo W.C., Toyama D., Motta B.P., Baviera A.M., Henrique-Silva F., dos Santos J.L, & Graminha M.A. (2021). Design, synthesis and biological evaluation of N-oxide derivatives with potent in vivo antileishmanial activity. PLoS ONE, 16(11), e0259008.

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Culturing PC3 Prostate Cancer Cells

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The human prostate cancer cell line PC3 was obtained from the American Type Culture Collection (ATCC^® CRL-1435™). The cells were routinely grown in Roswell Park Memorial Institute (RPMI) essential medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (LGC Biotechnology, São Paulo, Brazil) and 1% penicillin–streptomycin (Sigma Aldrich, St. Louis, MO, USA) in a humidified 5% CO₂ atmosphere at 37 °C. The cells were cultured to 70–100% confluence. The compounds were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) to produce a 10 mM stock solution, and the compounds were dissolved in RPMI at different concentrations. The DMSO concentration did not exceed 0.1% and did not have any effect.

Guerra F.S., Rodrigues D.A., Fraga C.A, & Fernandes P.D. (2021). Novel Single Inhibitor of HDAC6/8 and Dual Inhibitor of PI3K/HDAC6 as Potential Alternative Treatments for Prostate Cancer. Pharmaceuticals, 14(5), 387.

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In Vitro Study of Benign Breast Tumor Cells

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For the in vitro study, cells from the benign breast tumor cell line E-20 were graciously provided by Professor Dr. Heidge Fukumasu from the Laboratory of Comparative and Translational Oncology at the University of São Paulo (USP). Cells were cultured in high-glucose Dubelcco’s modified Eagle medium (DMEM) obtained from Gibco™, New York, NY, USA. The culture medium was further supplemented with 10% fetal bovine serum sourced from LGC Biotecnologia, São Paulo, Brazil and 1% penicillin/streptomycin solution from LGC Biotechnology, São Paulo, Brazil. Cultured cells were incubated in a controlled environment at 37 °C with 5% CO₂. Upon reaching 80% confluence, the cells were divided into treatment groups as follows (Table 2).

Procópio de Oliveira C., Frigieri B.M., Fukumasu H., Chuffa L.G., Novais A.A, & Zuccari D.A. (2023). Potential Protective Role of Melatonin in Benign Mammary Cells Reprogrammed by Extracellular Vesicles from Malignant Cells. Biomedicines, 11(10), 2837.

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Cytotoxicity Evaluation of Nanoparticles on Murine Macrophages

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Murine macrophage lineage J774.16 was also used to assess the cytoxicity of the nanoparticles. Macrophages were maintained in DMEM medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Fetal Bovine Serum, LGC Biotecnologia, Cotia, Sao Paulo, Brazil). First, 180 μL of DMEM supplemented with 10% FBS, containing nanoparticles with different amounts of P10 ranging from 400 μg to 0.19 μg were distributed in 96-well plates. Then, 20 µL of a suspension containing 5 × 10⁴ cells/mL was added to each well.
After 24, 48 and 72 h of incubation with the nanoparticles, 20 μL of 5 mg/mL MTT was added to each well and incubated at 37 °C and 5% CO₂ for 4 h. The supernatants were then discarded and 100 μL of DMSO (dimethylsulfoxide, Sigma-Aldrich, St. Louis, MO, USA) was added to each well. Then, the absorbance of the supernatants was measured at 540 nm using a plate spectrophotometer reader. The percentage of live cells was calculated using the following Equation (3),

% of live cells = \frac{(100 \times Av)}{ADMEM}

where: Av: Absorbance of sample, ADMEM: Average absorbance of samples treated with DMEM (control).

Rodrigues Dos Santos Junior S., Kelley Lopes da Silva F., Santos Dias L., Oliveira Souza A.C., Valdemir de Araujo M., Buffoni Roque da Silva L., Travassos L.R., Correa Amaral A, & P. Taborda C. (2020). Intranasal Vaccine Using P10 Peptide Complexed within Chitosan Polymeric Nanoparticles as Experimental Therapy for Paracoccidioidomycosis in Murine Model. Journal of Fungi, 6(3), 160.

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Murine Bone Marrow Derived Dendritic Cells and Macrophages

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Bone marrow from femurs and tibiae of C57BL/6 mice were obtained according to the protocol established by Inaba et al., (19 (link)). For differentiation in DCs, the cells were cultured in RPMI 1640 medium (LGC, biotechnology, Brazil) supplemented with 10% (v/v) fetal bovine serum (LGC, biotechnology, Brazil) 1% (v/v) penicillin/streptomycin (Gibco, USA), 30 ng/ml of GM-CSF and 15 ng/ml of IL-4 (Invitrogen, USA). The culture medium was changed on the third and fifth days. On day 8, differentiated dendritic cells were obtained. For differentiation in Mφ, the cells were cultured in R20/30 medium (RPMI 1640, 20% (v/v) fetal bovine serum and 30% (v/v) supernatant of L929 cells) (20 (link)). The medium was changed on the fourth day and, on the seventh day, macrophages were obtained. All cells were incubated in a humidified CO₂ incubator at 37°C. The cell phenotype was confirmed by BD FACS Fortessa flow cytometer as described below.

Kischkel B., Boniche-Alfaro C., Menezes I.D., Rossi S.A., Angeli C.B., de Almeida S.R., Palmisano G., Lopes-Bezerra L., Nosanchuk J.D, & Taborda C.P. (2021). Immunoproteomic and Immunopeptidomic Analyses of Histoplasma capsulatum Reveal Promiscuous and Conserved Epitopes Among Fungi With Vaccine Potential. Frontiers in Immunology, 12, 764501.

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Quantification of Antibody Response to Ovalbumin

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After euthanasia, serum was collected to determine OVA-specific IgG1, IgG2a, and IgE antibodies, the plates were coated overnight at 4 °C with ovalbumin in a coating buffer solution. The plates were washed three times with washing buffer (PBS containing 0.05% Tween-20) and blocked with assay diluent (PBS containing 10% heat inactivated fetal bovine serum (LGC Biotecnologia, Cotia, São Paulo, Brazil) for 1 h. The serum samples from the mice were diluted in the test diluent and added to the plates after three more washes. After 2 h of incubation at room temperature (RT), the plates were washed five times, and diluted anti-IgG1, anti-IgG2a and anti-IgE detection antibodies were added to the plate. The plates were incubated for 1 h at room temperature and washed. The Substrate Reagent Set was added, and the plates were left for 30 min in the dark. The colorimetric reaction was interrupted by the addition of 2 N H₂SO₄. The absorbance was acquired at 450 nm in a Biotek Synergy HT microplate reader and was analyzed by Gen 5 software (BioTek). The values of the absorbance samples were considered after subtracting the values from the wells incubated with only fresh mouse serum at the same dilutions. The data are shown in optical density (OD) units [21 (link)].

Nascimento C.M., Casaro M.C., Perez E.R., Ribeiro W.R., Mayer M.P., Ishikawa K.H., Lino-dos-Santos-Franco A., Pereira J.N, & Ferreira C.M. (2023). Experimental allergic airway inflammation impacts gut homeostasis in mice. Heliyon, 9(6), e16429.

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