Fetal calf serum (fcs)

Manufactured by Merck Group

Sourced in United States, Germany, United Kingdom, France, Switzerland, Austria, Japan, Italy, Australia, Belgium, Hungary, Macao, China, Sweden, Denmark, Ireland, Canada, India, Israel, Netherlands, Poland

FCS is a highly sensitive and versatile instrument used for the analysis and characterization of microscopic particles and molecules in liquid samples. It operates on the principle of fluorescence correlation spectroscopy, allowing for the detection and quantification of various analytes, including proteins, nucleic acids, and nanoparticles, with high precision and sensitivity.

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Fetal calf serum (FCS 10%; Calf serum

2 058 protocols using fetal calf serum (fcs)

Cell Culture Maintenance Protocols

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K562, RAJI, 8866, C1R and 721.221 cells were maintained in RPMI (Sigma-Aldrich) supplemented with 10% fetal calf serum (Sigma-Aldrich), 2 mM glutamine (Biological Industries (BI)), 1 mM sodium pyruvate (BI), 1× nonessential amino acids (BI), 100 U/ml penicillin (BI), 0.1 mg/ml streptomycin (BI).
RKO, T-47D, and A549 cells were maintained in DMEM (Sigma-Aldrich) supplemented with 10% fetal calf serum (Sigma-Aldrich), 2 mM glutamine (Biological Industries (BI)), 1 mM sodium pyruvate (BI), 1× nonessential amino acids (BI), 100 U/ml penicillin (BI), 0.1 mg/ml streptomycin (BI).
NK-92 cells were maintained in two different media: RPMI (Sigma-Aldrich) supplemented with 10% fetal calf serum (Sigma-Aldrich), 2 mM glutamine (Biological Industries (BI)), 1 mM sodium pyruvate (BI), 1× nonessential amino acids (BI), 100 U/ml penicillin (BI), 0.1 mg/ml streptomycin (BI), and 200U/ml IL-2 (PeproTech); or MEM-alpha (BI) supplemented with 12.5% fetal calf serum (Sigma-Aldrich), 12.5% Horse serum (BI), 2 mM glutamine (BI), 1 mM sodium pyruvate (BI), 1× nonessential amino acids (BI), 100 U/ml penicillin (BI), 0.1 mg/ml streptomycin (BI), 200 U/ml IL-2 (PeproTech), 0.2 mM myoinositol, 0.02 mM folic acid, 0.1mM beta-mercaptoethanol, 0.2% Ribonucleosides and Deoxyribonucleosides for MEM-Alpha.

Kotzur R., Duev-Cohen A., Kol I., Reches A., Mandelboim O, & Stein N. (2022). NK-92 cells retain vitality and functionality when grown in standard cell culture conditions. PLoS ONE, 17(3), e0264897.

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Cell Culture Protocols for LEDGFKD and Control Cells

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All cells were grown in a humidified atmosphere containing 5% CO₂ at 37°C. HeLaP4 310 LEDGF/p75 depleted cells ([46 ], further referred to as LEDGF_KD cells) were grown in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO-BRL, Merelbeke, Belgium) supplemented with 5% v/v heat inactivated fetal calf serum (FCS; Sigma-Aldrich, Bornem, Belgium), 0.005% w/v gentamicin (GIBCO), 0.05% w/v geneticin (GIBCO) and 0.01% w/v zeocin (Life Technologies, Ghent, Belgium). These cells are monoclonal LEDGF_KD cells, derived from HeLaP4 cells (gift from P. Charneau, Institut Pasteur, Paris, France). HelaP4 cells were grown on DMEM (GIBCO) supplemented with 5% v/v heat inactivated fetal calf serum (FCS; Sigma-Aldrich), 0.005% w/v gentamicin (GIBCO) & 0.05% w/v geneticin (GIBCO). HEK 293T cells (gift from O. Danos, Evry, France) were cultured in DMEM medium (GIBCO) with 8% v/v heat inactivated FCS (Sigma-Aldrich) and 0.005% w/v gentamicin (GIBCO). SupT1 cells were cultured in Roswell Park Memorial Institutes medium (RPMI, GIBCO-BRL, Merelbeke, Belgium) supplemented with 10% v/v heat inactivated fetal calf serum FCS (Sigma-Aldrich, Bornem,Belgium) and 0.005% w/v gentamicin (GIBCO). Nalm pre-B cells were cultured in RPMI (GIBCO) with 10% v/v heat inactivated FCS (Sigma-Aldrich) and 0.005% w/v gentamicin (GIBCO).

Vranckx L.S., Demeulemeester J., Debyser Z, & Gijsbers R. (2016). Towards a Safer, More Randomized Lentiviral Vector Integration Profile Exploring Artificial LEDGF Chimeras. PLoS ONE, 11(10), e0164167.

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Prostate Cancer and Mesenchymal Stem Cell Co-culture

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Human dual-color variant PC3 prostate cancer cells expressing H2B-eGFP and DsRed2 were from Anticancer; luciferase-expressing PC3 cells were provided by Dr. Gary Gallick, UT MD Anderson Cancer Center. Cells were maintained in DMEM (Corning), 10% fetal calf serum (Sigma), penicillin and streptomycin (both 100 μg/ml, Sigma). Human C4–2B cells (provided by Dr. Timothy Thompson, UT MD Anderson Cancer Center) expressing H2B/mCherry and LifeAct-GFP were cultured in RPMI (Corning), 10% fetal calf serum (Sigma), penicillin and streptomycin (both 100 μg/ml, Sigma) and 1% HEPES. The identity of tumor cell lines was verified by Short Tandem Repeat DNA profiling (Characterized Cell Line Core Facility, M.D. Anderson Cancer Center). ASC52telo telomerase reverse transcriptase immortalized adipose tissue derived mesenchymal stem cells (hMSCs, ATCC) were maintained in Minimum Essential Medium (MEM1X, Corning), supplemented with 17% fetal calf serum, vitamins (Sigma), non-essential amino acids (Sigma), sodium pyruvate (Gibco), penicillin and streptomycin (both 100 μg/ml, Sigma). To induce osteoblastic differentiation, hMSCs were cultured in osteogenic medium (DMEM 1X, supplemented with 10% calf serum, penicillin and streptomycin, 50 μg/ml L-ascorbic acid, 10 mM β-glycerophosphate, 0.1 μM dexamethasone from Sigma).

Paindelli C., Navone N., Logothetis C.J., Friedl P, & Dondossola E. (2019). Engineered Bone for Probing Organotypic Growth and Therapy Response of Prostate Cancer Tumoroids in vitro. Biomaterials, 197, 296-304.

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Cell Culture Conditions for Cell Lines

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U2OS cells were grown in Dulbecco’s modified Eagle’s medium with Glutamax (Gibco, Life technologies, UK) containing 10% fetal calf serum (Sigma-Aldrich, Saint-Quentin-Fallavier, France) and 1% zellshield (Minerva Biolabs, Berlin, Germany). These cells were transfected with JetOptimus (polyplus transfection, Illkirch, France).
Calu-6 cells were grown in Roswell Park Memorial Institute medium 1640 with Glutamax, containing 10% fetal calf serum (Sigma-Aldrich) and 1% zellshield (Minerva Biolabs).
16HBE14o- cells were grown in Minimum Essential Medium Eagle with Glutamax (Gibco), containing 10% fetal calf serum (Sigma-Aldrich) and 1% zellshield (Minerva Biolabs).

Carrard J., Ratajczak F., Elsens J., Leroy C., Kong R., Geoffroy L., Comte A., Fournet G., Joseph B., Li X., Moebs-Sanchez S, & Lejeune F. (2023). Identifying Potent Nonsense-Mediated mRNA Decay Inhibitors with a Novel Screening System. Biomedicines, 11(10), 2801.

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Cell Culture and Western Blotting Protocol for Lymphoma Cell Lines

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Cell lines were cultured in RPMI (Invitrogen) with 10% fetal calf serum (Sigma-Aldrich), except for SU-DHL-4 and SU-DHL-6 which were cultured in RPMI with 20% fetal calf serum, and OCI-Ly10 which was cultured in Iscove‘s modified Dulbecco medium with 10% fetal calf serum. All cell lines were maintained at 37 °C. SU-DHL-4, SU-DHL-6, Karpas422, DOHH-2 and WSU-DLCL2 were purchased from DSZM, Pfeiffer was purchased from ATCC and OCI-Ly10 and HBL-1 were gifts from the Weng lab (BCCRC) to the LCR lab. All cell lines were authenticated by STR profiling. SU-DHL-6. HBL-1 and WSU-DLCL2 were not mycoplasma tested but all others tested negative.
Western Blotting was performed as described^{26 (link)} using the Rabbit Polyclonal IκBζ Antibody (TA336346) (Origene) (dilution 1:500) and the Histone H3 Antibody #9715 (Cell Signaling) (dilution 1:1000). Un-cropped western blot is shown in Supplementary Figure 11.

Arthur S.E., Jiang A., Grande B.M., Alcaide M., Cojocaru R., Rushton C.K., Mottok A., Hilton L.K., Lat P.K., Zhao E.Y., Culibrk L., Ennishi D., Jessa S., Chong L., Thomas N., Pararajalingam P., Meissner B., Boyle M., Davidson J., Bushell K.R., Lai D., Farinha P., Slack G.W., Morin G.B., Shah S., Sen D., Jones S.J., Mungall A.J., Gascoyne R.D., Audas T.E., Unrau P., Marra M.A., Connors J.M., Steidl C., Scott D.W, & Morin R.D. (2018). Genome-wide discovery of somatic regulatory variants in diffuse large B-cell lymphoma. Nature Communications, 9, 4001.

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Cell Culture Conditions for Various Cell Lines

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SIRC (Cornea, ATCC CCL-60), RAB-9 (Skin, ATCC CRL-1414), RK13 (Kidney, ATCC CCL-37) and RL-33 (Lung, tebu-bio JCRB0131) cell lines were cultured in monolayers in MEM GlutaMAX™ (Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS, Sigma Aldrich) and 1% Penicillin/Streptomycin (P/S, Sigma Aldrich). 55D1 (B-cell line, (61 (link)); kind gift of Dr. Katherine L. Knight) and RL-5 (T-cell line; 62 ) were cultured in RPMI 1640 GlutaMAX™ Medium (Gibco) supplemented with 10% FCS (Sigma Aldrich) and 1% P/S (Sigma Aldrich). HEK293T cells (Kidney, human, DSMZ ACC 635) were cultured in monolayers in DMEM GlutaMAX™ (Gibco) supplemented with 10% FCS (Sigma Aldrich) and 1% P/S (Sigma Aldrich). All cells were cultured at 37°C and 5% CO₂ and 90% humidity.

Schelle L., Côrte-Real J.V., Fayyaz S., del Pozo Ben A., Shnipova M., Petersen M., Lotke R., Menon B., Matzek D., Pfaff L., Pinheiro A., Marques J.P., Melo-Ferreira J., Popper B., Esteves P.J., Sauter D., Abrantes J, & Baldauf H.M. (2024). Evolutionary and functional characterization of lagomorph guanylate-binding proteins: a story of gain and loss and shedding light on expression, localization and innate immunity-related functions. Frontiers in Immunology, 15, 1303089.

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Cell Culture Protocols for Pancreatic Cancer

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Human AsPC-1 and MIA PaCa-2 pancreas carcinoma cells were obtained from Prof. Dr. Ulrich Massing, University Hospital Freiburg, Germany, Clinic for Tumor Biology and their identity was confirmed by Short-tandem repeat profiling. AsPC-1 cells were grown in RPMI 1640 medium (PAN Biotech, Aidenbach, Germany) containing 10% (v/v) fetal calf serum (FCS, Sigma Aldrich, Steinheim, Germany), 100 U/mL penicillin, and 100 µg/mL streptomycin (PAN Biotech). MIA PaCa-2 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (PAN Biotech, Aidenbach, Germany) containing 10% (v/v) fetal calf serum (FCS, Sigma Aldrich, Steinheim, Germany), 100 U/mL penicillin, and 100 µg/mL streptomycin (PAN Biotech). Capan-2 cells were kindly obtained from Prof. Ingo Schmidt-Wolf, University Hospital Bonn, Germany, and cultivated in RPMI 1640 medium (PAN Biotech) containing 10% (v/v) fetal calf serum (FCS, Sigma Aldrich), 100 U/mL penicillin and 100 µg/mL streptomycin (PAN Biotech). All cell lines were incubated at 37 °C in a humidified atmosphere containing 5% (v/v) CO₂.
For subcultivation, cell lines were treated with trypsin/EDTA (5 g/L trypsin; 0.2 g/L EDTA × 4 Na, Sigma Aldrich) for 5 min at 37 °C. Mycoplasma check was routinely performed every month and cell identity was validated using a STR profile analysis.

Haschemi R., Gockel L.M., Bendas G, & Schlesinger M. (2021). A Combined Activity of Thrombin and P-Selectin Is Essential for Platelet Activation by Pancreatic Cancer Cells. International Journal of Molecular Sciences, 22(7), 3323.

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Intestinal Mucosal Immune Response Assay

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Intestinal explants were used to test the reactivity of the mucosal immune system [31 (link),32 (link)]. Adapted from Chatelais et al. [33 (link)], 5 cm segments of jej-PP, jej-LP and il-PP were rinsed with PBS containing 1% dithiothreitol (DTT, Sigma) and 1% FCS (Sigma) and then placed in a solution made with 74% PBS, 25% DMEM (Sigma), 1% FCS, 5 µg/mL gentamicin (Sigma) and 1.25 µg/mL amphotericin B (Sigma) for immediate explant culture. Jejunal and ileal mucosa were then cut in small biopsies of 10–15 mg. Biopsies (two per well, weighing, in total, 20 to 35 mg) were cultured for 2 h under an atmosphere containing 5% CO₂ at 37 °C [33 (link)]. Explants were then transferred into the same solution as above but without FCS (replacement with BSA), and stimulated or not with 50 µg/mL lipopolysaccharides (LPS, TLR4 ligand; Sigma) or 1 µg/mL flagellin (TLR5 ligand; Invivogen, Toulouse, France) and incubated for 20 h under an atmosphere containing 5% CO₂ at 37 °C. Finally, supernatants were collected and frozen at −20 °C for later cytokine analysis, and the biopsies were harvested in 1 mL of Trizol reagent (Fisher Scientific, Illkirch, France) and frozen at −80 °C for RNA extraction.

Ferret-Bernard S., Le Normand L., Romé V., Le Bourgot C., Seeboth J., Savary G., Laurent F., Le Huërou-Luron I, & Guzylack-Piriou L. (2020). Maternal Supplementation of Food Ingredient (Prebiotic) or Food Contaminant (Mycotoxin) Influences Mucosal Immune System in Piglets. Nutrients, 12(7), 2115.

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Cultivation and Transfection of Cell Lines

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B16-F1 mouse melanoma cells (ATCC CRL-6323) were grown in DMEM (4.5 g l⁻¹ glucose; Invitrogen, Germany) with 10% FCS (PAA Laboratories, Austria) and 2 mM glutamine (Thermo Fisher Scientific), and transfected using peqFECT transfection reagent (PeqLab). Three micrograms DNA in total and 6 μl peqFECT reagent were used to transfect cells in 10 cm dishes overnight. NIH 3T3 fibroblasts (ATCC CRL-1658), Raw macrophages (ATCC TIB-71), J774 macrophages (ATCC TIB-67) and Rac1^−/− MEFs40 (link) were grown in DMEM (4.5 g l⁻¹ glucose), 10% FCS (Sigma), 2 mM L-glutamine, 1% non-essential amino acids and 1 mM sodium pyruvate. Vero (ATCC CCL-81) and MDCKI (ATCC CCL-34) epithelial cell lines were maintained in DMEM (4.5 g l⁻¹ glucose), 10% FCS (Sigma) and 2 mM L-glutamine. DU-145 (ATCC HTB-81) and LoVo (ATCC CCL-229) were cultivated in RPMI 1640 medium (Gibco), 10% FCS (Sigma), 2 mM L-glutamine, 1% non-essential amino acids and 1 mM sodium pyruvate. All cells were incubated at 37 °C in the presence of 7.5% CO₂.

Kage F., Winterhoff M., Dimchev V., Mueller J., Thalheim T., Freise A., Brühmann S., Kollasser J., Block J., Dimchev G., Geyer M., Schnittler H.J., Brakebusch C., Stradal T.E., Carlier M.F., Sixt M., Käs J., Faix J, & Rottner K. (2017). FMNL formins boost lamellipodial force generation. Nature Communications, 8, 14832.

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Cytotoxicity Evaluation of Extracted Samples

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The
extraction of test samples was performed according to ISO 10993-5
(2009)¹² and as described by Menz et al.^{8 (link)} by mixing 2.0 g of each test sample diluted in
PFO with 6.6 mL of the Dulbecco’s Modified Eagle’s Medium
(DMEM) extraction vehicle containing 10% fetal calf serum (FCS, Sigma-Aldrich)
at 37 °C for 24 h. The extraction of the test samples containing
the PFOA was performed in the absence of FCS, as recommended by the
ISO 10993-5 (2009)¹² for polar substances;
however, FCS was added to the DMEM vehicle before application to the
cells in the cytotoxicity test. After extraction, each sample underwent
centrifugation at 5000 rpm for 15 min: the water-insoluble phase was
discarded, and the aqueous phase was used for the cytotoxicity test
on the extracts.

Gatto C., Ruzza P., Giurgola L., Honisch C., Rossi O., Romano M.R., Ragazzi E, & D’Amato Tóthová J. (2022). Comparison of Perfluorocarbon Liquids Cytotoxicity Tests: Direct Contact Versus the Test on Liquid Extracts. ACS Omega, 8(1), 365-372.

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