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Glutmax

Manufactured by Thermo Fisher Scientific
Sourced in United States, China

GlutMAX is a laboratory instrument designed for the measurement of glutathione levels in biological samples. It provides a reliable and efficient method for quantifying this important antioxidant in a variety of cell and tissue types.

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34 protocols using glutmax

1

Culturing Human Melanoma and Control Cell Lines

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The human melanoma M285, M375 and M296, cell lines were a kind gift from Antoni Ribas (University of California, Los Angeles), and the M202, A375, M229, SKmel28 and SKmel5 cells were a kind gift from Randall T. Moon and Andy J. Chien (University of Washington, Seattle). Primary human melanocytes adult (HEMa-LP) were obtained from Gibco. Human embryonic kidney cells (HEK-293) and rhabdomyosarcoma cells (RD-1) were obtained from the Biomedical Research Centre (University of East Anglia, UK). Human melanoma cells were cultured as previously described [29 (link)]. HEMa-LP cells were cultured in Medium-254 (Gibco) with the addition of PMA-Free Human Melanocyte Growth Supplement-2 (HMGS-2; Gibco). HEK-293 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) + GlutMAX (Gibco) supplemented with 10% FBS, 1% L-glutamine and penicillin and streptomycin. RD-1 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) + GlutMAX (Gibco) supplemented with 10% FBS and penicillin and streptomycin. All cells were maintained at 37° C in a 5% CO2 air-humidified incubator, were routinely screened for mycoplasma and not cultured beyond passage 25.
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2

Hypoxic Neuron Culture and Treatment

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The mouse Neuro‐2a cells (N2a, ATCC® CCL131™) were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, 2311542) with 10% fetal bovine serum (FBS, Gibco, 10099141) and 1% penicillin/streptomycin (PS, Gibco, 15070063). Primary neurons were prepared from 18‐days old embryos as described previously.21 Briefly, freshly dissected cerebrums were dissociated and the neurons were seeded at a density of 9 × 105 cells/ml on poly‐d‐lysine‐coated (ST508, Beyotime) dishes in neurobasal medium (Gibco, 21103049) with 2% B27 (Gibco, 17504044), 1% Glutmax (Gibco, 35050079), and 1% PS. Cytarabine (10 μM/L) (Sigma, PHR1787) was applied at 48 h to kill the glia. Upon maturation (usually about 7 days), the neurons of the hypoxic group were cultured in 1% O215 (calculated with alveolar gas equation according to the altitude of 7000 m and the capillary blood flow rate and metabolism.22, 23 The normal concentration of oxygen in the brain is about 4.4%24), 5% CO2 at 37℃ in the incubator (Smartor118/118pro) for 4, 8, and 12 h, respectively. The concentration of FA incubation was 0.5 mM (in completed cultural medium) and last for 8 h. Neurons were incubated in CoQ10 (1 mM, in PBS) for 30 min before detection.
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3

Culturing Human Glioblastoma and Astrocytes

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Human GBM cell lines were purchased from Sigma-Aldrich (St. Louis, MO, USA) (U251) or ATCC (Manassas, VA, USA) (U118). U251 cells were cultured in GBM culture medium, which included MEM (Gibco, Gaithersburg, MD, USA), 0.2% penicillin/streptomycin (Gibco), 10% fetal bovine serum (FBS; Gibco), 1 mM sodium pyruvate (Gibco), 1% non-essential amino acids (Gibco), and 1× GlutMAX (Gibco). U118 cells were cultured in medium containing DMEM (Gibco), 10% FBS, and 1% penicillin/streptomycin.
Human astrocytes were purchased from ScienCell (San Diego, CA, USA). Human astrocytes were cultured in human astrocyte medium, which included DMEM/F12 (Gibco), 10% FBS, 3.5 mM glucose (Sigma-Aldrich), and 0.2% penicillin/streptomycin, supplemented with B27 (Gibco), N2 (Gibco), 10 ng/mL fibroblast growth factor 2 (Invitrogen, Carlsbad, CA, USA), and 10 ng/mL epidermal growth factor (Invitrogen).
For subcultures, the cells were trypsinized using 0.25% trypsin (Gibco) or TrypLE Select (Invitrogen), centrifuged for 5 min at 800 rpm, resuspended, and plated in corresponding culture medium with a split ratio of approximately 1:4. The cells were maintained at 37 °C in humidified air with 5% CO2.
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4

Culturing Primary Cortical Neurons

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Primary cortical neurons were prepared from neonatal (p0) C57BL/6J mice. Dissected cortices were digested with papain (2 mg/ml) for 30 min at 37 °C and digestion was stopped using 10% FBS. Cortical neurons (500 cells/mm2) were plated on poly-D-lysine pre-coated glass dishes in DMEM (Sigma-Aldrich) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco) for 4 hours. Afterwards, the medium was replaced with neurobasal medium A (Gibco) containing 1% penicillin-streptomycin, 1% GlutMax (Gibco) and 2% B27 (Gibco). The PALP imaging experiment was performed with primary neurons cultured for 2 days in vitro (DIV 2).
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5

Cell Culture and Compound Preparation Protocol

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293T cells were purchased from ATCC. HepG2 cells were obtained from Prof Aifu Lin (Zhejiang University, China) and Huh7 cells were obtained from Prof Xiaoyuan Lian (Zhejiang University, China). MIHA, Hep3B, HCC-LM3, PLC/PRF5, HLE and Sun387 cells were obtained from Prof Shusen Zheng (Zhejiang University, China). 293T, MIHA, HepG2, Hep3B, HCC-LM3, PLC/PRF5, HLE cells were cultured in DMEM (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (Gibco). Huh7 cells were maintained in DMEM (Gibco) with 10% fetal bovine serum and 1% GlutMAX (Gibco). Sun387 cells were cultured in RPMI 1640 (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (Gibco). Trichostatin A (TSA, MCE, New Jersey, USA), Nicotinamide (NAM, MCE) were dissolved in dimethyl sulfoxide (DMSO). Fluphenazine hydrochloride (FPZ, National Institutes for Food and Drug Control, Beijing, China), Ferric ammonium citrate (FAC, Sigma-Aldrich, St. Louis, MO) and Deferoxamine mesylate (DFO, MCE) were dissolved in phosphate buffer saline (PBS). Iron dextran (National Institutes for Food and Drug Control) was dissolved in 0.9% NaCl.
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6

Neural Stem Cell Proliferation and Differentiation

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Icariin (ICA) (purity > 98% by high-performance liquid chromatography) was purchased from Shaanxi Scidoor Hi-tech Biology Co. Ltd. (Xi'an, China). Growth medium was composed of serum-free conditioned Dulbecco's modified Eagle's medium/Ham's F-12 medium (DMEM/F12, Gibco) with 20 ng/ml EGF and 10 ng/ml bFGF (Gibco, USA). NSC proliferation medium was composed of DMEM/F12 and B27 (2%) supplemented with 20 ng/ml EGF + 20 ng/ml bFGF. Basic medium was composed of DMEM/F12 and B27 (2%). NSC differentiation medium was composed of DMEM/F12, B27 (2%), GlutMAX (1%), and FBS (1%) (Gibco, USA). All cell plates or covered glasses were precoated with poly-D-lysine (Sigma, USA).
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7

Maintenance of Pluripotent Stem Cells

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DMEM (high glucose, Hyclone), 1% N2 (GIBCO), 2% B27 (GIBCO), 1% GlutMax (GIBCO), 1% NEAA (GIBCO), 1% sodium pyruvate (GIBCO) 3 mM CHIR99021, 1 mM PD0325901 and 1000 U LIF.
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8

Maintenance of Pluripotent Stem Cells

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DMEM (high glucose, Hyclone), 1% N2 (GIBCO), 2% B27 (GIBCO), 1% GlutMax (GIBCO), 1% NEAA (GIBCO), 1% sodium pyruvate (GIBCO) 3 mM CHIR99021, 1 mM PD0325901 and 1000 U LIF.
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9

Isolation and Culture of Mouse Embryonic Fibroblasts

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Mouse embryonic fibroblasts (MEFs) carrying the Oct4-GFP reporter were isolated from E13.5 embryos. MEFs and Plate-E cells were cultured in DMEM (Hyclone, high glucose) supplement with 10% FBS (Natocor), GlutaMAX (Gibco), and non-essential amino acids (Gibco). Mouse iPSCs and ESCs were maintained in serum-containing FBS medium (DMEM supplement with 10% FBS (Gibco), 1× GlutMAX, 1× NEAA, 1× Sodium pyruvate (Gibco), 0.1 mM β-mercaptoethanol (Gibco), PD03325901 (1 μM, In house-synthesized), CHIR99021 (3 μM, In house-synthesized), 1000 units/ml LIF (Millipore). All of the cell lines have been confirmed as mycoplasma contamination free with kit from Lonza (LT07–318).
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10

Isolation of Hippocampal Neurons from Rats

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Hippocampal neurons were isolated from embryonic day 18 (E18) Sprague–Dawley rats and cultured as described previously.30 (link) Briefly, embryos were decapitated after cleaning with 75% alcohol. The hippocampus without meninges was carefully extracted from the brain under a microscope (Olympus, Tokyo, Japan) and transferred to an ice-cold buffer composed of 127 mM NaCl, 1.7 mM NaH2PO4, 5 mM KCl, 2.05 mM KH2PO4, 10nM D-glucose, and 100 U mL−1 penicillin/streptomycin (pH 7.4). Shredded hippocampus tissues were dissociated using the Neuron Isolation Enzyme Kit (with papain) (Thermo Fisher, MA, USA), and then incubated at 37 °C for 10 min. The tissue solution was obtained following a wash with Hank’s Balanced Salt Solution (Gibco, MA, USA), collected on a 100-mesh grid, and centrifuged at 1000 rpm for 5 min. After centrifugation, the supernatant was discarded. Primary rat neurons were resuspended in a minimum essential medium composed of 1% GLUT-MAX and 2% B-27 Supplement (both from Gibco, MA, USA). A sufficient volume of the cell suspension was transferred to a petri dish containing poly-L-lysine-coated coverslips for culture at 37 °C and 5% CO2 in a sterile incubator.
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