The murine feeder-independent ESC line CGR8 was cultured in gelatin-coated flasks in DMEM (Gibco) supplemented with 100 U/mL LIF (ESGRO-Millipore), 2 mM L-glutamine (Gibco), 2 mM non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Gibco) and 15% heat-inactivated HyClone FBS (GE Healthcare Life Sciences). For EB formation, cells were trypsinized and diluted in Iscove's modified Dulbecco's medium (Gibco) supplemented with 2 mM L-glutamine (Gibco), 2 mM non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Gibco), and 20% heat-inactivated HyClone FBS (GE Healthcare Life Sciences), to a final concentration of 1,000 cells/20 μL. EBs were cultured without LIF as hanging drops for 2 days and subsequently collected and cultured in suspension for 6 additional days.
Hyclone fbs
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
HyClone FBS is a high-quality fetal bovine serum (FBS) product from GE Healthcare. It is a complex mixture of proteins, hormones, and other factors that support the growth and proliferation of various cell types in cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Cell staining buffer, by BioLegend (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Pen strep, by Thermo Fisher Scientific (3 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (3 mentions)
Bright glo luciferase assay system, by Promega (3 mentions)
Atplite luminescence assay kit, by PerkinElmer (3 mentions)
Mouse anti firefly luciferase antibody, by Santa Cruz Biotechnology (3 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
11995 dmem, by Thermo Fisher Scientific (2 mentions)
Transit lt1 transfection reagent, by Mirus Bio (2 mentions)
Viafect transfection reagent, by Promega (2 mentions)
Hek293t, by American Type Culture Collection (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
X vivo 15, by Lonza (2 mentions)
43 protocols using hyclone fbs
1
Feeder-independent Murine ESC Culture
2
OVCA Cell Line Maintenance and Transfection
3
Immunoblotting of Nanoluc and β-Tubulin
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SW982 human synovial sarcoma cells were cultured in Roswell Park Memorial Institute (RPMI 1640; Gibco) medium supplemented with 10% HyCloneTM FBS (GE Healthcare) and 1% penicillin-streptomycin (Gibco), at 37°C with 5% CO2. The cells were seeded in 6-well plates at least 6 h prior to transfection using the ViaFectTM transfection reagent (Promega). One day after transfection, cells were harvested and lysed. Total cell lysates were resolved on a denaturing 10% SDS-PAGE gel, and blotted onto a PVDF membrane using the Trans-Blot Turbo Transfer system (Bio-Rad). Samples were then probed with (1) Nanoluc antibody (R&D Systems; MAB100261), resuspended to 306 μg/mL and diluted to 1:200 with 5% milk in TBST, and (2) β-tubulin antibody (Cell Signaling, # 2146), which was supplied in a concentration of 100 μg/mL, and diluted to 1:1000 with the 5% bovine serum albumin (BSA) in TBST. The samples were then probed with the appropriate secondary antibody: (1) α-mouse secondary antibody diluted to 1:2500 in blocking solution for Nanoluc, and (2) α-rabbit secondary antibody diluted to 1:2500 in blocking solution for β-tubulin. The membrane was incubated in substrate ECL HRP substrate (Advansta), and chemiluminescent signals were obtained using the ImageQuant LAS 4000 mini (GE Healthcare Life Sciences).
4
Culturing and Transfecting Human Synovial Sarcoma Cells
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SW982 human synovial sarcoma cells were cultured in Roswell Park Memorial Institute (RPMI 1640; Gibco) medium supplemented with 10% HyCloneTM FBS (GE Healthcare) and 1% penicillin-streptomycin (Gibco), at 37°C with 5% CO2. The cells were seeded in 6-well plates at least 6 h prior to transfection using the ViaFectTM transfection reagent (Promega). One day after transfection, the media was removed and replaced with RPMI 1640 containing puromycin dihydrochloride (Thermo Fisher Scientific) diluted to a final concentration of 5 μg/mL. The medium containing the selection antibiotic was changed every 3–4 days for 2 weeks, after which the puromycin concentration was lowered to 2 μg/mL for culture maintenance.
5
Engineered Cell Lines for Research
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HEK293T (ATCC) and MV4-11 (ATCC) were cultured in media per the supplier’s recommendation. Expi293 (Thermo Fisher Scientific) was cultured in media per the supplier’s recommendation. U87-EGFR is a kind gift from Cellectis SA (Paris, France). U87-EGFR was derived from the parental cell line, U87MG (ATCC) by first knocking out endogenous EGFR using Transcription Activator-Like Effector Nucleases (TALEN), and then stably overexpressing full-length human EGFR via lentiviral transduction. X-VIVOTM 15 was obtained from Lonza, rhIL-2 from Miltenyi Biotech, human serum AB from Seralab, human T-activator CD3/CD28 from Thermo Fisher Scientific, MACS® LD-column from Miltenyi Biotech, Hyclone FBS from GE Healthcare Life Sciences, Ficoll-Paque PLUS from GE Healthcare Life Sciences and Pan T cell Isolation Kit, human from Miltenyi Biotech. Methotrexate disodium salt and leucovorin calcium were obtained from Alfa Aesar and Sigma, respectively.
6
Comprehensive Reagents for Cell Culture Experiments
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The following items were purchased from Gibco: MEM (11095), DMEM (11965092), HI FBS (14000), Pen/Strep (15140). TrypLE (12604013), PBS −/− (w/o Ca2+ or Mg2+) (10010049), Trypsin-EDTA (25300–054). Hyclone FBS (SH30071.03) was purchased from GE Healthcare. The following items were purchased from ATCC: EMEM (30–2003), Vero-E6 (CRL-1586, RRID:CVCL_0574), HeLa (CCL-2, RRID:CVCL_0030), HEK293T (CRL-3216, RRID:CVCL_0063). Huh-7.5 cells were a gift from the Tang Lab at FSU. The following items were purchased from Invitrogen: Live Cell Imaging Buffer (A14291DJ), LysoTracker Deep Red (L12492), goat-anti-mouse AlexaFluor-647 (A-21242, RRID:AB_2535811), HCS Cell Mask Green (H32714), Hoechst 33342 (H3570). LC3B primary rabbit antibody (3868S, RRID:AB_2137707) was purchased from Cell Signaling Technologies. Cell Staining Buffer (420201) was purchased from BioLegend. The following items were purchased from Corning: 384-well plates (3764 BC), BioCoat 384-well poly-d-Lysine coated plates (354663 BC), Amphotericin B (30–003-CF). 100% Methanol (34860) was purchased from Sigma-Aldrich. Calpain Inhibitor IV (208724) was purchased from CalbioChem.
7
Isolation and Culture of Ascitic Tumor Cells
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Chemo-naïve ascitic fluid was centrifuged at 500xg for 10 min at 4°C and cell pellets pooled in HBSS media. Red blood cells were removed using a red blood cell lysis buffer (Miltenyi Biotec) as per the manufacturer’s instructions. Tumor cells were counted and plated at 1 million cells in two collagen-coated 75 cm2 flasks and cultured in OCMI media containing 5% Hyclone™ FBS (GE Healthcare). All cultures were initially incubated for 2-4 day at 37°C in a humidified 5% CO2, 5% O2 atmosphere and media was replaced every 3-4 day. Upon cell attachment, stromal cells were separated from the mixed sample using 0.05% trypsin-EDTA and plated in gelatin-coated 75 cm2 flasks in OCMI media containing 5% FBS. Once tumor cells reach 95% confluency, cells were passaged using 0.25% Trypsin-EDTA, centrifuged in DMEM containing 20% FBS and re-plated at a 1:2 ratio. For long-term storage, cells were frozen in Bambanker™ (Wako pure chemical).
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Nutrient-Rich Cell Culture Medium
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Nutrient Mixture F12-Hams: Medium 199 (50:50) mixed media containing 5% FBS (Life Science group) or 5% Hyclone™ FBS (GE Healthcare); 2 mM glutamine; 100 U/ml penicillin; 100 U/ml streptomycin; 10 mM HEPES at pH 7.4; 20 μg/ml insulin; 0.01 μg/ml EGF; 0.5 μg/ml hydrocortisone; 10 μg/ml transferrin; 0.2 pg/ml Tridothyronine; 5 μg/ml o-phosphoryl ethanolamine; 8 ng/ml selenious acid; 0.5 ng/ml 17β-oestradiol; 5 μg/ml all trans retinoic acid; 1.75 μg/ml hypoxanthine; 0.05 μg/ml lipoic acid; 0.05 μg/ml cholesterol; 0.012 μg/ml ascorbic acid; 0.003 μg/ml α-tocopherol phosphate; 0.025 μg/ml calciferol; 3.5 μg/ml choline chloride; 0.33 μg/ml folic acid; 0.35 μg/ml vitamin B12; 0.08 μg/ml thamine HCL; 4.5 μg/ml i-inositol; 0.075 μg/ml uracil; 0.125 μg/ml ribose; 0.0125 μg/ml para-aminobenzioic acid; 1.25 mg/ml BSA; 0.085 μg/ml xanthine and 25 ng/ml cholera toxin (all from Sigma).
9
Culturing Airway Epithelial Cells
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We passaged the primary NHBE cells (passage 1) three times before differentiating them (passage 4) into a pseudostratified epithelium. For each passage, the cells were grown in a 100-mm culture dish (Corning, Inc.) precoated with PurCol (Cell Systems, 5005). The cells were maintained in airway epithelial cell (AEC) growth medium (PromoCell; C20601) containing AEC supplements (PromoCell), 2% penicillin/streptomycin (Thermo Fisher Scientific, 15140122), and 1% amphotericin B (Thermo Fisher Scientific, 15290026) (complete AEC medium) at 37°C in an incubator with 5% CO2. The cells were grown to 90% confluence, and the medium was changed every other day. Confluent monolayers of cells were dissociated with 5 mL of TrypLE (Thermo Fisher Scientific, 12604021) and pelleted, and one-third of the cells were reseeded in a culture dish containing complete AEC medium for passaging. A549 cells were grown in F-12 medium (Thermo Fisher Scientific, 11765054) with 10% HyClone FBS (GE Healthcare, SH3007103), 2% penicillin/streptomycin, and 1% amphotericin B.
10
Murine Macrophage RAW264.7 Cell Culture
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The murine macrophage RAW264.7 cell line was purchased from the American Type Culture Collection (ATCC). The cells were cultured in Dulbecco modified Eagle medium (DMEM) (HyClone; GE Healthcare Life Sciences) containing 10% fetal bovine serum (HyClone FBS; GE Healthcare Life Sciences) and 1% (v/v) penicillin–streptomycin solution (HyClone Penicillin–Streptomycin solution; GE Healthcare Life Sciences). Cells were maintained at 37°C in a 5% CO2 incubator.
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