All animal experiments were approved by the animal welfare ethics committee of the University of Tokyo. Dissociated primary hippocampal cultures were prepared from E16.5 ICR mouse embryos of either sex as described previously with minor modifications (Okabe et al., 1999 (link)). First, hippocampi were treated with trypsin (Gibco) and DNase (Sigma). Then, they were mechanically dissociated and suspended in MEM containing B18 supplement, L-glutamine (Gibco), and 5% FCS (Equitech-Bio). After preparation of cell suspensions, they were plated onto a glass-bottom dishes (MatTek) coated with poly-L-lysine (Sigma). To prevent glial cell proliferation, 5 μM ara-C (Sigma) was added to cultures 2 d after plating.
Fetal calf serum (fcs)
Manufactured by Equitech-Bio
Sourced in United States, Japan
Fetal calf serum is a complex mixture of proteins, growth factors, and other nutrients derived from the blood of bovine fetuses. It is commonly used in cell culture media to support the growth and maintenance of various cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Amphotericin b, by Thermo Fisher Scientific (6 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Insulin, by Merck Group (3 mentions)
Hydrocortisone, by Merck Group (3 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Dmem f12k, by Merck Group (2 mentions)
F12 ham dmem f12k, by Merck Group (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by GE Healthcare (2 mentions)
Streptomycin sulfate, by Thermo Fisher Scientific (2 mentions)
Igepal ca 930, by Merck Group (2 mentions)
Express one step sybr greener universal kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Testosterone, by Steraloids (2 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Ara c, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Dnase, by Merck Group (1 mentions)
16 protocols using fetal calf serum (fcs)
1
Primary Mouse Hippocampal Culture
2
Calcium Imaging of Exogenous H1 Receptor
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HeLa cells (ATCC) were seeded, at the densities indicated, on 12-mm diameter cover slips in Minimum Essential Medium Alpha Medium (Invitrogen, Gaithersburg, MD) containing 10% fetal calf serum (FCS, Equitech-Bio, Ingram, TX). Cells were transfected with plasmids using Lipofectin (Invitrogen) and cultured for 48–72 h to allow expression of exogenous cDNA. To identify HeLa cells expressing exogenous H1 receptor by calcium imaging, pME-H1 was co-transfected with pEGFP-C1. For FRET imaging pCFP-PHD and pYFP-PHD were co-transfected, with or without pME-H1. HEK293 cells (ATCC) were seeded in Dulbecco’s Modified Eagle’s Medium (DMEM Asahi Technoglass, Funabashi, Japan) containing 10% fetal calf serum (FCS).
3
Isolation and Culture of Human Naïve B Cells
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Peripheral blood mononuclear cells (PBMCs) were separated from heparinized whole blood by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). B cells were purified using Human B Cell Isolation Kit II (Miltenyi Biotec), and naïve B cells were isolated using Human Naïve B Cell Isolation Kit (Miltenyi Biotec). The ethics committee of the University of Tokyo Hospital approved this study (No. 10154 and G3582). All subjects provided written informed consent, and the study was conducted in accordance with relevant guidelines.
Unless otherwise indicated, cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% FCS (Equitech Bio), 100 μg/ml L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 50 μM 2-ME (Sigma). In some experiments, cells were cultured in X-VIVO15 (Lonza) to exclude the effect of TGF-β in FCS.
TGF-β1 and β3 (R&D) were used at 1 ng/ml unless otherwise indicated. IL-21 (PeproTech), IL-4 (R&D), soluble CD40L (PeproTech), and CpG-ODN2006 (Enzo Life Sciences) were used at 50 ng/ml, 100U/ml, 2 μg/ml, and 6 μg/ml respectively, and BCR stimulation was induced using goat anti-human IgA + IgG + IgM (H+L) (Jackson ImmunoResearch) at 2.5 μg/ml.
Unless otherwise indicated, cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% FCS (Equitech Bio), 100 μg/ml L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 50 μM 2-ME (Sigma). In some experiments, cells were cultured in X-VIVO15 (Lonza) to exclude the effect of TGF-β in FCS.
TGF-β1 and β3 (R&D) were used at 1 ng/ml unless otherwise indicated. IL-21 (PeproTech), IL-4 (R&D), soluble CD40L (PeproTech), and CpG-ODN2006 (Enzo Life Sciences) were used at 50 ng/ml, 100U/ml, 2 μg/ml, and 6 μg/ml respectively, and BCR stimulation was induced using goat anti-human IgA + IgG + IgM (H+L) (Jackson ImmunoResearch) at 2.5 μg/ml.
4
Silica-Induced Macrophage and Epithelial Cell Responses
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The mouse macrophage-like cell line RAW264.7 (derived from BALB/c mice) from the American Type Culture Collection (ATCC; Rockville, MD, USA), and bronchial epithelial cells, transformed human bronchial epithelial cells 16HBE, kindly provided by D.C. Gruenert (Gene Therapy Center, University of California, CA, USA) were used [26 (link)]. The cells were cultured in DMEM (Sigma) supplemented with 10% FCS (Equitech-Bio, Kerrville, TX, USA), 1% penicillin, and 1% streptomycin (Sigma) at 37 °C in 5% CO2. The cells were cultured to subconfluence in 6-well plates (Sumitomo, Osaka, Japan), then rendered quiescent in medium containing 0.5% FCS for another 1 day, followed by exposure of 0.1 or 0.5 mg/ml of sterilized silica for 24 h. In some experiments, the cells were pretreated with one of the following agents before silica treatment: 1 h with 50 or 100 μM of the ERK inhibitor, U0126 (Cell Signaling Technology, Danvers, MA, USA), 1 h with 25 mM or 50 mM tetramethylthiourea (TMTU; MP Biomedicals, Santa Ana, CA, USA), 16 h with 5 or 10 μM bilirubin (Sigma), 1 h with 20 or 50 μM RuCO (CO-releasing molecule; Sigma), 1 h with 100 or 200 μM hemin, or 1 h with 100 or 200 μM ZnPP. The cells were collected either 2–6 or 8–24 h after silica exposure and evaluated for ERK activation or HO-1 induction, respectively, in RAW264.7 or 16HBE cells.
5
Culturing Osteosarcoma and MDCK Cell Lines
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The osteosarcoma D-17 (ATCC® CCL-183™) cell line was purchased from Cientifica Senna (Ciudad de México, México) and MDCK (ATCC® CCL-34™) cell line was kindly donated by Laura Cobos-Marín (UNAM, México). Both cell lines were routinely cultured according to the supplier’s recommendations. Cells were grown in Dulbecco’s modified Eagle’s medium/nutrient F-12 Ham (DMEM/F12K, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum (Equitech Bio, Kerrville, TX, USA), 100 U/mL penicillin/streptomycin (Gibco), and 1 µg/mL amphotericin B (Invitrogen) at 37 °C in a humidified atmosphere with 5% CO2. All experiments were performed using cell lines maintained at low passage numbers (5–10 passages). Cisplatin, carboplatin, and doxorubicin were purchased from Sigma and work solutions were prepared in Dulbecco’s modified Eagle’s medium/nutrient F-12 Ham.
6
Bovine Mammary Epithelial Cell Isolation
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bMECs were isolated from the alveolar tissue of the udders of healthy lactating cows (slaughtered for meat production) as described (10 (link)), which were obtained at the slaughterhouse. This protocol was approved by the ethical committee for animal welfare from Universidad Michoacana de San Nicolás de Hidalgo. Cells from passages two to eight were used in all of the experiments. bMECs were cultured in growth medium (GM) composed by DMEM medium/nutrient mixture F12 Ham (DMEM/F12K, Sigma) and supplemented with 10% fetal calf serum (Equitech Bio), 10 μg/ml insulin (Sigma), 5 μg/ml hydrocortisone (Sigma), 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml amphotericin B (Invitrogen). bMECs were grown in a 5% CO2 atmosphere at 37°C.
7
Isolation of Peripheral Blood Mononuclear Cells
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Peripheral blood was drawn from three healthy Japanese adults, collected in Venoject II vacuum blood collection tubes (heparinized blood collection tubes) (Terumo, Tokyo, Japan), and transferred to 50 mL centrifuge tubes in the presence of 2 mM ethylenediaminetetraacetic acid (EDTA). After two-fold dilution with phosphate buffered saline (PBS), peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation on Percoll (d = 1.077) (GE Healthcare, Tokyo, Japan), at 500–800 g for 30 min. The remaining red blood cells were further lysed by ACK lysing solution (0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM Na2EDTA at pH 7.4). If required, the debris not dispersed after suspension of the cell pellet with PBS was carefully removed using a pipette. PBMCs were washed with PBS and suspended in culture medium [RPMI1640 (Nacalai Tesque, Kyoto, Japan) with 10% fetal calf serum (Equitech-Bio Inc., Kerrville, TX, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Nacalai Tesque)], at a density of 1 × 106 cells/mL. One hundred microliters of PBMCs were seeded on each well of a 96-well cell culture plate (TPP, Trasadingen, Switzerland), and precultured in a humidified 5% CO2 incubator at 37°C for 4 h.
8
Isolation and Culture of Bovine Mammary Epithelial Cells
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bMECs were isolated from the alveolar tissue of the udders of healthy lactating cows as previously described [13 (link)]. Cells from passages 2–8 were used in all of the experiments. The bMECs were cultured in growth medium (GM) that was composed of a DMEM medium/nutrient mixture F12 Ham (DMEM/F12K, Sigma) supplemented with 10% fetal calf serum (Equitech Bio), 10 μg/mL insulin (Sigma), 5 μg/mL hydrocortisone (Sigma), 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 μg/mL amphotericin B (Invitrogen). The cells were grown in a 5% CO2 atmosphere at 37°C. To perform the E2 and/or S. aureus challenge, polarized monolayers of bMECs (dishes covered with 6–10 μg/cm2 rat-tail type I collagen, Sigma) were cultured in serum-free DMEM/F12K without phenol red (Sigma) and antibiotics (incomplete medium) for 24 h, and then they were treated with the hormone and/or infected with the bacteria.
9
Extracellular and Intracellular ATP Measurement in 3T3-L1 Adipocytes
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Materials. Dulbecco's modified Eagle's medium (DMEM) was purchased from Wako (Osaka, Japan); fetal calf serum from Equitech-Bio Inc. (Cotton Gin Lane, Kerrville, TX); mouse monoclonal antibodies against bovine mitochondrial F 1 F 0 -ATP synthase α (Vα) and β (Vβ) subunits from Molecular Probes (Eugene, OR); NF279 from Cosmo Bio Co. LTD. (Tokyo, Japan); MRS2500 from Tocris Bioscience (Bristol, UK); apyrase from SIGMA (St. Louis, MO).
Cell culture and differentiation. Murine 3T3-L1 preadipocytes were maintained in DMEM with 10% (v/ v) fetal calf serum and each sample in a humidified atmosphere of 95% air and 5% CO 2 at 37°C. Adipocyte differentiation was performed as described previously (2) . Determination of ATP. Extracellular ATP was determined using the cellular ATP assay reagent (Toyo-B-net, Tokyo). Briefly, 3T3-L1 adipocytes (day 16) cultured on the 96-well plates were incubated with each sample for 48 h, and cell-culture supernatants were collected. An aliquot of 100 μL of each supernatant was added to the 96-well plate, and then 100 μL of ATP assay reagent was added. After 1 min of shaking and 10 min of incubation at 23°C, luminescence was detected with a microplate reader. Intracellular ATP was determined using the same cells (after their supernatants were collected) and reagents cellular ATP contributes to the cell-surface F 1 F 0 -ATP synthase-mediated TG accumulation.
Cell culture and differentiation. Murine 3T3-L1 preadipocytes were maintained in DMEM with 10% (v/ v) fetal calf serum and each sample in a humidified atmosphere of 95% air and 5% CO 2 at 37°C. Adipocyte differentiation was performed as described previously (2) . Determination of ATP. Extracellular ATP was determined using the cellular ATP assay reagent (Toyo-B-net, Tokyo). Briefly, 3T3-L1 adipocytes (day 16) cultured on the 96-well plates were incubated with each sample for 48 h, and cell-culture supernatants were collected. An aliquot of 100 μL of each supernatant was added to the 96-well plate, and then 100 μL of ATP assay reagent was added. After 1 min of shaking and 10 min of incubation at 23°C, luminescence was detected with a microplate reader. Intracellular ATP was determined using the same cells (after their supernatants were collected) and reagents cellular ATP contributes to the cell-surface F 1 F 0 -ATP synthase-mediated TG accumulation.
10
SCID and AT Cell Culture
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SCID cells (SC3VA2) [32 (link)] and AT cells (AT5BIVA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (Equitech-Bio, INC. Kerrville, TX, USA).
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