All cell lines were maintained at 37 °C in a humidified atmosphere at 5% CO2, unless otherwise indicated in figure legends or method details. Human ES-2 cells (cat. no. CRL-1978) were purchased from ATCC and routinely maintained in McCoy’s 5A (Gibco) supplemented with 10% FBS (Lonza). FreestyleTM 293-F cells (cat. no. p/n51-0029) were obtained from Invitrogen and routinely maintained in 293 FreeStyleTM medium. The CHO-K1 cell line (cat. no. DSMZ ACC110) was obtained from DSMZ and routinely maintained in DMEM/F12 (Gibco) supplemented with L-Glutamine (Gibco) and 10% FBS (Lonza). MDA-MB-231 cells (cat. no. CRM-HTB-26) were obtained from ATCC and routinely maintained in DMEM high glucose (Gibco) supplemented with 100 mM sodium pyruvate (Gibco), MEM non-essential amino acids (Gibco), and 10% FBS (Lonza). BxPC-3 cells (cat. no. CRL-1687) were obtained from ATCC and routinely maintained in RPMI-1640 (Gibco) supplemented with 10% FBS (Lonza). A549-A2-ESO tumor cells are derived from a human lung cancer cell line and express the cancer-testis antigen NY-ESO-1 in the appropriate HLA context and were cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS), 100U/ml penicillin, 100 µg/ml streptomycin sulfate, and 1% L-Glutamine (complete cell culture medium).
Fetal bovine serum (fbs)
Manufactured by Lonza
Sourced in Switzerland, United States, Belgium, France, Germany, Spain, United Kingdom, Italy, Japan, Austria, Macao, China, Poland, Australia, India, Netherlands, Holy See (Vatican City State)
FBS (Fetal Bovine Serum) is a cell culture supplement derived from the blood of bovine fetuses. It provides a rich source of proteins, growth factors, and other essential nutrients required for the optimal growth and proliferation of cells in vitro. FBS is a widely used component in cell culture media, supporting the maintenance and propagation of a variety of cell types.
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Lab products found in correlation
L glutamine, by Lonza (157 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (147 mentions)
Streptomycin, by Thermo Fisher Scientific (139 mentions)
Penicillin streptomycin, by Lonza (133 mentions)
Penicillin, by Thermo Fisher Scientific (132 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (119 mentions)
Penicillin, by Lonza (115 mentions)
Streptomycin, by Lonza (114 mentions)
Rpmi 1640, by Lonza (97 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (88 mentions)
L glutamine, by Thermo Fisher Scientific (87 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (81 mentions)
Rpmi 1640, by Thermo Fisher Scientific (52 mentions)
Penicillin, by Merck Group (51 mentions)
Streptomycin, by Merck Group (51 mentions)
Rpmi 1640 medium, by Lonza (51 mentions)
L glutamine, by Merck Group (46 mentions)
Trypsin edta, by Lonza (46 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (39 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (36 mentions)
1 566 protocols using fetal bovine serum (fbs)
1
Maintenance of Common Cell Lines
2
Culturing Pancreatic Cancer Cell Lines
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MIA PaCa-2, human PC cells, were cultured in DMEM (Lonza) containing L-Glutamine 2 mM, 10% heat-inactivated fetal bovine serum (FBS; Lonza) and 2,5% heat inactivated horse serum (HS; Lonza). PANC-1, human pancreatic epithelioid carcinoma cells, were kept in DMEM containing L-Glutamine 2 mM and 10% heat-inactivated fetal bovine serum (FBS; Lonza). BxPC-3, human pancreatic adenocarcinoma cells, were cultured in RPMI 1640 (Lonza) containing 10% heat-inactivated fetal bovine serum (FBS; Lonza). CAPAN-2, human PDAC cells, were kept in McCoy’s 5a Medium Modified (Lonza) with 10% heat-inactivated fetal bovine serum (FBS; Lonza). All the media were supplemented with antibiotics (10000 U/ml penicillin and 10 mg/ml streptomycin; Lonza). Cell lines were purchased from ATCC (Rockville, USA) and were grown at 37°C in 5% CO2 -95% air humidified atmosphere.
3
Isolation of Mononuclear Cells from Ileum and MLNs
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To isolate mononuclear cells (MNCs) from the ileum and MLNs, tissues were minced and digested in RPMI-1640 medium (Lonza) supplemented with 2 mg/mL collagenase/dispase (Roche Diagnostics) and 5% fetal bovine serum (FBS) (Lonza) at 37 °C with periodic agitation for 40 minutes. The cell suspension was sequentially passed through 100-μm and 40-μm cell strainers (BD Biosciences) and centrifuged at 500 X g for 10 minutes. The cell pellet was resuspended in RPMI-1640 medium (Lonza) containing 5% FBS (Lonza) and layered on an OptiPrep (Stemcell Technologies) density gradient. Following centrifugation at 750 X g for 30 minutes with the brake disengaged, the light density fraction was collected and washed twice with ice-cold PBS. The washed cells were resuspended in RPMI-1640 medium (Lonza) supplemented with 5% FBS (Lonza) and 2% antibiotics-antimycotics (Lonza). Cell viability and counts were determined by trypan blue dye exclusion using a hemocytometer.
4
Culturing Human and Murine Cell Lines
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Human hepatic stellate cells (LX2 cells), provided by Prof. Scott Friedman (Mount Sinai Hospital, New York, NY, USA), were cultured in DMEM‐Glutamax medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Lonza, Verviers, Belgium) and antibiotics (50 U/ml Penicillin and 50 μg/ml streptomycin, Sigma, St. Louis, MO, USA). Murine RAW 264.7 macrophages and human THP1 monocytes, obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Lonza) supplemented with 10% FBS (Lonza), 2 mM L‐glutamine (Sigma) and antibiotics. Human HepG2 cells (ATCC) were cultured in DMEM‐high glucose medium (Lonza) supplemented with 10% FBS and antibiotics. All the cell lines used in this study were authenticated with STR profiling and were tested regularly for the absence of mycoplasma contamination by PCR.
5
TGF-β1-induced Myofibroblast Differentiation
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Human GFs were cultured in DMEM (Lonza) supplemented with 10% FBS (Lonza) and 0.1% gentamicin sulfate (10 mg/ml; Euroclone) at 37°C in 5% CO2 atmosphere. Cells between the fourth and tenth passages were used for the following experiments. At Passage 6 cells were seeded overnight with medium containing 1% of FBS (Lonza) and then were treated with or without TGF-β1 for 24, 48, and 72 h. Medium was removed 24 h after seeding and fibroblasts were incubated for 24, 48, and 72 h with DMEM plus with 1% FBS (Lonza) serum alone or supplemented with 10 ng/ml TGF-β1 (S.I.A.L.). Myofibroblasts phenotype was confirmed by flow cytometry, confocal microscopy, and Western blot for α-SMA, Vimentin, E-cadherin, β-catenin, and Smad 2/3. Once the obtained cell differentiation cells were collected to perform all experiments, the untreated cells were used as negative controls (CTRL).
6
Culturing AML Cell Lines and Primary Blasts
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AML cell lines HL-60 (ACC-3)[30 (link)], KG-1 (ACC-14)[31 (link)], MonoMac-1 (ACC-252)[32 (link)] and Kasumi-1 (ACC-220)[33 (link)] were obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) and the human stroma cell line HS-5 was purchased from ATCC (American Type Culture Collection). Experiments were performed within 6 months after receipt or resuscitation. AML cell lines were cultured in complete RPMI medium (PAA laboratories) supplemented with fetal bovine serum (Lonza), sodium pyruvate (Lonza) and/or non-essential amino acids (Lonza) according to manufacturers' recommendations. HS-5 cell line was cultured in complete DMEM medium (PAA laboratories) supplemented with 10% fetal bovine serum (Lonza). Primary AML blasts were cultured in IMDM (PAA laboratories) supplemented with 3% heat-inactivated fetal bovine serum (Lonza), 1x BIT (StemCell Technologies), 5 ng/ml IL3 (Peprotech), sodium pyruvate (Lonza) and β-mercaptoethanol (Sigma).
7
Evaluation of PIM and FLT3 Inhibitors
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Human PMBL cells Karpas1106P and U2940 and L-428 cHL cells were maintained in RPMI 1640 medium supplemented with 20% fetal bovine serum (Lonza Group AG, Basel, Switzerland). SUP-HD1 cHL cells were grown in McCoy's 5A medium supplemented with 20% fetal bovine serum (Lonza Group AG). B-cell acute lymphoblastic leukemia SEM cell line was cultured in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum (Lonza Group AG). The dual pan-PIM-FMS-like tyrosine kinase 3 (FLT3) inhibitor MEN1703 was provided by Menarini Ricerche (Pomezia, Italy), and the pan-PIM inhibitor PIM447 was purchased from Selleckchem (Houston, TX). All chemicals were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO). In vehicle-control experiments, final DMSO concentrations was 0.5% (control for a 5-mmol/L dose).
8
Generation of Stable Cell Lines for Protein Expression
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HeLa and RPE1 FlpIn cells were respectively grown in DMEM and DMEM/F12 supplemented with 8% FBS (Lonza), penicillin/streptomycin (50 μg ml−1), Ultra-glutamine (Sigma; 2 mM), blasticidin (4 μg ml−1) and hygromycin for HeLa (200 μg ml−1) or puromycin for RPE1 (1.6 μg ml−1). 293Ts were grown in DMEM supplemented with 8% FBS (Lonza), penicillin/streptomycin (50 μg ml−1) and Ultra-glutamine (Sigma; 2 mM). Plasmids were transfected using Fugene HD (Roche) for HeLa or Lypofectamin LTX (Invitrogen) for RPE1 according to the manufacturer's instructions. To generate stably integrated HeLa and RPE1 FlpIn cell lines, pCDNA5-constructs were co-transfected with pOG44 recombinase in a 1:9 for HeLa and 1:5 ratio for RPE1 (ref. 55 (link)). Constructs were expressed by addition of 1 μg ml−1 doxycycline for 24 h siHEC1 (custom; Thermo Fisher Scientific; 5′-CCCUGGGUCGUGUCAGGAA-3′) and siGAPDH (Thermo Fisher Scientific; D-001830-01-50) were transfected using HiPerfect (Qiagen) according to manufacturer's instructions.
Cells expressing RFP-MAD2 were obtained through lentiviral transduction and subsequent selection with puromycin (1.6 μg ml−1).
Cells expressing RFP-MAD2 were obtained through lentiviral transduction and subsequent selection with puromycin (1.6 μg ml−1).
9
Culture of Rat and Mouse Insulinoma Cells
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INS1E rat insulinoma cells (provided by Dr. Bruno Guigas, LUMC, Leiden, The Netherlands and Dr. Pierre Maechler, University Medical Center, Geneva, Switzerland) were cultured in RMPI (Gibco) with 2.05 mM Glutamax (Invitrogen, Bleiswijk, The Netherlands) supplemented with 5% (v/v) fetal bovine serum (FBS, Lonza, Verviers, Belgium), 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco, Bleiswijk, The Netherlands), 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 1 mM sodium pyruvate, and 50 µM freshly added β-mercaptoethanol (Gibco) at 37 °C and 5% CO2. Mouse insulinoma MIN6-B1 cells (provided by Dr. Phillipe Halban, University Medical Center, Geneva, Switzerland) were cultured in DMEM (Gibco) supplemented with 10% (v/v) FBS (Lonza), 100 U/mL penicillin and 100 mg/mL streptomycin, and 70 µM freshly added β-mercaptoethanol (Gibco) (37 °C, 5% CO2).
10
Cell Culture Maintenance Protocols
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Vero (ATCC CCL81), HEK-293 (ATCC CRL1573) and HEK-293T cells (ATCC CRL3216) were maintained at 37°C, 5% CO2 in Dulbecco Modified Eagle’s Medium (DMEM, Thermo Fisher scientific, Montigny-le-Bretonneux, France) supplemented with 5% fetal bovine serum (FBS, Lonza), 1 mM sodium pyruvate, penicillin (1 U/mL)/streptomycin (1 μg/mL) and 2 mM L-glutamine. BHK-21 cells (ATCC CCL10) were maintained at 37°C in Dulbecco Modified Eagle’s Medium (DMEM, Thermo Fisher scientific) supplemented with 10% fetal bovine serum (FBS, Lonza, France) and penicillin (1 U/mL)/streptomycin (1 μg/mL). C6/36 cells (ATCC CRL1660) were maintained at 28°C, in Leibowitz L-15 Medium supplemented with 1 mM sodium pyruvate, penicillin (1 U/mL)/streptomycin (1 μg/mL), 1 mL non-essential amino acids, and 2 mM L-glutamine (Thermo Fisher Scientific, France).
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