Human CSs were derived as previously described 12 . In brief, heart tissues were minced into small pieces about 1–2 mm3, then washed with PBS and digested with collagenase solution for 15 min. (Sigma‐Aldrich, St. Louis, MO, USA), the tissue fragments were cultured as “cardiac explants” on plate coated with 0.25 mg/ml fibronectin (BD Biosciences, San Jose, CA, USA) in Iscove's modified Dulbecco's medium (IMDM; Invitrogen, Carlsbad, CA, USA). The IMDM media was supplemented with 20% foetal bovine serum (FBS; Corning, Corning, NY, USA), 0.5% Gentamicin (Gibco, Life Technologies, Durham, CA, USA), 0.1 mM 2‐mercaptoethanol (Invitrogen) and 1% L‐glutamine (Invitrogen). In about 1–2 weeks, a layer of stromal‐like flat cells, and phase‐bright round cells, emerged from the cardiac explant with phase‐bright cells over them. These cardiac explant‐derived cells were harvested using TryPEL Select (Gibco) and then seeded at a density of 2 × 104 cells/ml in poly‐d‐lysine‐coated flaks for cardiosphere formation. In about 3–5 days, explant‐derived cells spontaneously aggregated into cardiospheres. The morphology of cardiospheres was checked with a Nikon phase contrast microscope.
Foetal bovine serum fbs
Manufactured by Corning
Sourced in United States
Foetal bovine serum (FBS) is a cell culture media supplement derived from the blood of bovine fetuses. It provides a rich source of proteins, growth factors, and other nutrients that support the growth and proliferation of various cell types in in vitro cell culture applications.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Corning (3 mentions)
Streptomycin, by Corning (3 mentions)
Sw1990 l 15, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
L glutamine streptomycin, by Corning (2 mentions)
Insulin, by Thermo Fisher Scientific (2 mentions)
Trypsin edta, by Lonza (2 mentions)
Insulin, by Merck Group (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Rpmi 1640, by Corning (2 mentions)
L glutamine, by Corning (2 mentions)
Dulbecco s modified eagle medium dmem, by Corning (2 mentions)
Trypsin edta, by Corning (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
27 protocols using foetal bovine serum fbs
1
Cardiac Explant-Derived Cardiosphere Formation
2
Immortalized Human Cardiomyocytes Characterization
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Immortalized human cardiomyocytes cells were used as a cardiac model and purchased from Applied Biological Materials Inc. (abm). They derived from the ventricular tissue of 62 old years male. The culture media was composed of Dulbecco’s modified Eagle’s medium/Ham’s F12 50/50 mix containing 10% Foetal bovine serum (FBS), 100 U/ml penicillin, 100 U/ml streptomycin, 2 mM glutamine (Corning, Manassas, VA, USA) and it was replaced day by day. The cells (used at passage 4–8) were subcultured by 0.25% trypsin–EDTA (Corning, Manassas, VA, USA) enzymatic digestion. For cardiomyocytes characterization, cells were seeded in 10% FBS supplemented DMEM F12 at a seeding density of 2.5 × 104 cells/cm2. 24 h after seeding, 10% FBS supplemented DMEM F12 was replaced with the culture medium supplemented with 1% of FBS, which was changed every day until the sixth day. Then, human cardiomyocytes were exposed to NSAIDs of interest.
3
PAAD Cell Lines Culture Conditions
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Human PAAD cell lines SW1990, PANC1 and BXPC3 (Chinese Acad emy of Sciences Cell Bank, China), HEK-293T and human pancreatic ductal cell HPDE6C7 cell lines (Beina Culture Collection, China) were obtained. SW1990 (L-15, Gibco, USA), BXPC3 (RPMI 1640, HyClone, USA), PANC1, HPDE6C7 and HEK-293T (DMEM, HyClone, USA) were cul tured with 10% foetal bovine serum (FBS; Corning, USA) at 37°C with 5% CO 2 .
4
Human OSCC Cell Line Establishment
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Human OSCC cell line, HOC313, established from oral floor, was kindly provided by the Department of Oral and Maxillofacial Surgery, Graduate School of Medical Science, Kanazawa University (Ishikawa, Japan). Another human OSCC cell line, SAS, established from a human squamous cell carcinoma of the tongue,22 (link),23 was obtained from the RIKEN BioResource Center (Ibaraki, Japan). The human OSCC cell line, OSC-19, was obtained from Kanazawa University (Ishikawa, Japan). OSC-19 cells were transfected with the pmR-ZsGreen1 (Takara Bio, Shiga, Japan) vector, and the cell line that stably expresses green fluorescent protein (GFP), OSC-19-GFP, was established. The human keratinocyte line, HaCaT, was kindly provided by Dr. Shirasuna, at Kyushu University (Fukuoka, Japan). The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma) supplemented with 10% foetal bovine serum (FBS; Corning) and maintained under a humidified 5% CO2 atmosphere at 37 °C.
5
HMGB1-induced activation of immune cells
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PBMCs, LD-PBMCs, HD-PBMCs, NK, NK-T cells, monocytes, and YTS cells were grown in Serum Containing Medium (SCM) RPMI 1640 plus 10% Foetal Bovine Serum (FBS) (Corning, New York, NJ, USA) and penicillin/streptomycin 5% solution (Thermofisher Scientific, Waltham, MA, USA) and activated with IL-2 (Preprotech, London, UK) (200 U/mL) for 72 h at 37 °C and 5% CO2, and, afterwards, incubated with human HMGB1 (Sigma Aldrich, Saint Louis, MO, USA) (2 μg/mL) for 48 h at 37 °C, as already reported [68 (link)]. TLR2 and TLR4 inhibition were performed using an anti TLR2 blocking antibody (10 μg/mL) and TAK242 (100nM) (Invivogen, San Diego, CA, USA), as already reported [69 (link)]. Inhibitors were added 24 h before HMGB1 treatment, during IL-2 incubation.
6
Breast Cancer Cell Culture Conditions
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MDA-MB-231 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies, CA, USA) and HCC1143 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, Carlsbad, CA, USA). Media was supplemented with 10% foetal bovine serum (FBS; Corning, Corning, NY, USA) and 1% penicillin-streptomycin solution (Life Technologies). MCF-12A cells were grown in MammoCult™ media supplemented with 5% FBS (Corning, NY, USA), 10% MammoCult™ proliferation supplement, 0.5% hydrocortisone and 0.2% heparin (all from Stem Cell Technologies, Vancouver, BC, Canada). MDA-MB-231-Luc cells were cultured in L-15 media supplemented with 15% FBS and 1% penicillin-streptomycin (Life Technologies, Carlsbad, CA, USA). A week before the animal experiment, the cells were harvested, washed in Dulbecco’s phosphate-buffered saline (DPBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and grown as mammospheres in MammoCult™ media (described below).
7
NRF2 Inhibition in Cancer Cell Lines
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The cell lines used in this study were: human SKBR3 (breast cancer, carrying R175H p53 mutation), T98MG (glioblastoma, carrying M237I p53 mutation), MCF7 breast cancer, U87 glioblastoma, HCT116 and RKO colon cancer cell lines (all carrying wild-type p53), and HCT116 p53 null. The cells were cultured in either DMEM (Dulbecco modified Eagle’s medium) (Life Technologies-Invitrogen), or RPM1–1640 (Life Technologies-Invitrogen), with 10% heat-inactivated foetal bovine serum (FBS) (Corning, NY, USA, #35–079) and L-glutamine/streptomycin (100 μg/mL) (Corning, NY, USA, #30–002), in 5% CO2 at 37 °C. They were all mycoplasma negative. The NRF2 inhibitor Brusatol [47 (link)] (Sigma-Aldrich) was used at 100 nM, as previously reported [48 (link)].
8
Sulforaphane Modulation of Cancer Cell Lines
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Human colon cancer HCT116, HCT116-p53−/− (kindly provided by Prof. Bert Volgelstein, Johns Hopkins University, Baltimore, MD, USA), colon cancer RKO and the human lung cancer H1299 (p53 null) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies-Invitrogen, Eggenstein, Germany), containing 10% heat-inactivated Foetal Bovine Serum (FBS) (Corning, NY, USA) and L-glutamine/streptomycin (100 µg/mL) (Corning, NY, USA) in a culture incubator with 5% CO2 at 37 °C in humidified atmosphere. They underwent routine testing to ensure that they were mycoplasm negative. The activator of NRF2, that is, D,L-Sulforaphane (1-isothiocyanato-4-methylsulfinylbutane (SFN) (Sigma-Aldrich, St. Louise, MO, USA) [26 (link)] was dissolved in DMSO and used at various concentrations (1, 2, 5 µM) for 24 h, alone or in combination with drugs; chemotherapeutic drug cisplatin (CDDP) (Pharmachemie BV, The Netherlands) was added to the cell culture at 5 μg/mL, as previously reported [22 (link)]; and ZnCl2 (Sigma-Aldrich) was dissolved in dH2O2 and used at 100 μM [27 (link)].
9
Cell Culture and Hemisynthesis Protocol
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For cell culture, Dulbecco modified Eagles minimal essential medium (DMEM)/HAM's F12 (F-12 Nutrient Medium) Glutamax, insulin, transferrin and selenium were obtained from Thermo Fisher (Waltham, MA, USA). Foetal bovine serum (FBS) was purchased from Corning (New York, NY, USA). Antibiotics (streptomycin and penicillin) and trypsin/EDTA were obtained from Lonza (Basel, Switzerland). The PA was purchased from Sigma-Aldrich (St Louis, MO, USA).
Concerning hemisynthesis and UPLC-DAD-MS analysis, acetobromo-α-Dglucuronic acid methyl-ester, pyridine and K 2 CO 3 were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France); and ultra-pure methanol, ethanol, acetonitrile, ethyl acetate and formic acid were purchased from Fisher Scientific. Milli-Q water used for these experiments was produced in the laboratory using Purelab Ultra System (Elga Lab Water, High Wycombe, UK).
Concerning hemisynthesis and UPLC-DAD-MS analysis, acetobromo-α-Dglucuronic acid methyl-ester, pyridine and K 2 CO 3 were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France); and ultra-pure methanol, ethanol, acetonitrile, ethyl acetate and formic acid were purchased from Fisher Scientific. Milli-Q water used for these experiments was produced in the laboratory using Purelab Ultra System (Elga Lab Water, High Wycombe, UK).
10
Analytical Standards of Bacopaside I and II
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The analytical standards bacopaside I (CAS No. 382148-47-2, 89.6% purity by HPLC, Lot no. 00002002-T17H) and bacopaside II (CAS No. 382146-66-9, 98% purity HPLC, Lot Number: 00002002-T17H), derived from the medicinal herb bacopa monnieri, were obtained from ChromaDex (Irvine, CA, USA), solubilised in methanol at 10 mM and 1.5 mM stock solutions, respectively, and stored at −20 °C. Cell lines MDA-MB-231, T47D, MCF7 and BT-474 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were maintained in complete medium, either in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) for MDA-MB-231, MCF7 and BT-474 or Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, Carlsbad, CA, USA) for T47D, containing 10% foetal bovine serum (FBS) (Corning, NY, USA), 200 U/mL of penicillin, 200 μg/mL of streptomycin (pen strep; Life Technologies, Carlsbad, CA, USA) and 2 mM L-alanyl-L-glutamine dipeptide (GlutaMAX Supplement; Life Technologies, Carlsbad, CA, USA). Cells were grown under standard culture conditions at 37° C with 5% CO2 in air and used within 4 passages.
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