L glutamine

Manufactured by Genesee Scientific

L-glutamine is a non-essential amino acid that is commonly found in the human body. It serves as a key component in various metabolic processes and is important for maintaining cellular function.

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7 protocols using l glutamine

Nonsense-Mediated Decay Inhibition in LCLs

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Human LCLs were obtained from the Coriell Institute. The cells were grown in suspension in RPMI 1640 medium (Genesee Scientific) supplemented with 15% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin and maintained at 37°C with 5% CO₂. To test the impact of NMD inhibition, two million cells of each LCL (GM19204, GM18508, GM19193, GM19238, GM12878, and S003659) were grown overnight and subsequently treated with 100 µg/ml of emetine (Sigma) for 7 h (Noensie and Dietz 2001 (link)). Parallel cultures were left untreated and grown at standard conditions. HeLa cells were grown in Dulbecco's Modified Eagle Medium, High Glucose, with L-Glutamine (Genesee Scientific) supplemented with 10% fetal bovine serum (Gibco, Life Technologies), penicillin (100 U/ml), and streptomycin (100 µg/ml) (Gibco, Life Technologies) at 37°C with 5% CO₂.

Shew C.J., Carmona-Mora P., Soto D.C., Mastoras M., Roberts E., Rosas J., Jagannathan D., Kaya G., O’Geen H, & Dennis M.Y. (2021). Diverse Molecular Mechanisms Contribute to Differential Expression of Human Duplicated Genes. Molecular Biology and Evolution, 38(8), 3060-3077.

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Endothelial Cell Line BSO Treatments

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Endothelial cell lines (mouse aortic endothelial cells; MAECs and mouse vena cava endothelial cells; VCECs) were cultured in Dulbecco’s modified Eagle Media-DMEM (Caisson labs, UT) with 10% Fetal bovine serum-FBS (Caisson labs, UT), added L-glutamine (Genesee Scientific, CA), penicillin and streptomycin (Genesee Scientific, CA) at 37^oC in 5% CO₂. Cells were grown to confluence followed by BSO (Sigma, MO) treatments of increasing concentrations (0, 5, 25, 50, 75, and 100 μM) for 19hrs in starvation media (with 0.5% FBS).

Shrestha B., Prasai P.K., Kaskas A.M., Khanna A., Letchuman V., Letchuman S., Alexander J.S., Orr A.W., Woolard M.D, & Pattillo C.B. (2018). Differential Arterial and Venous Endothelial Redox Responses to Oxidative Stress. Microcirculation (New York, N.Y. : 1994), 25(7), e12486.

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Procurement and Characterization of HGSOC Tissues

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Human high grade serous ovarian carcinoma (HGSOC) tissues were procured under protocols approved by the Committee for the Protection of Human Subjects at Dartmouth–Hitchcock Medical Center (no. 17702); under a protocol approved by the H. Lee Moffitt Cancer Center (MCC no. 18974). Patients were female in gender and their ages ranged from 23-85 with a median value of 57. Informed consent was obtained from all them. Tumor chunks were either freshly dissociated and cryopreserved; or freshly dissociated, sorted (CD45⁻EpCAM⁺ primary HGSOC cells) and cultured continuously in R10 medium (RPMI-1640, 10% FBS, penicillin/Streptomycin (100 μg/mL Lonza), l-glutamine (2 mM, Genesee Scientific), sodium pyruvate (0.5 mM) (ThermoScientific)) until they adhered to establish primary HGSOC cell lines. Established cell lines were used for subsequent experiments.
Ovarian tumor tissue microarrays (TMAs) were obtained from 3 different resources: the Tissue Core at Moffitt Cancer Center (MCC cohort; approval MCC no. 50264) which include 93 HGSOC and some control tissues; TriStar Technology Group, LLC (TA-1966, Rockville, MD), which include 51 HGSOC; and US BioMax, Inc. (Ov401sur; Derwood, MD) with 8 HGSOC.

Anadon C.M., Yu X., Hanggi K., Biswas S., Chaurio R., Martin A., Payne K.K., Mandal G., Innamarato P., Harro C.M., Mine J., Sprenger K., Cortina C., Powers J.J., Costich T.L., Perez B.A., Gatenbee C.D., Prabhakaran S., Marchion D., Heemskerk M.H., Curiel T.J., Anderson A.R., Wenham R.M., Rodriguez P.C, & Conejo-Garcia J.R. (2022). Ovarian cancer immunogenicity is governed by a narrow subset of progenitor tissue-resident memory T-cells. Cancer cell, 40(5), 545-557.e13.

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Cell culture and synchronization protocols

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Table S2 lists all reagents and tools used in this study. HeLa cells (ATCC) were grown in DMEM/Ham's F-12 with L-Glutamine (Genesee Scientific), U2OS (ATCC) and HCT116 cells were grown in McCoy's 5A (Gibco), with 10% FBS and 5% CO₂ at 37 °C. Detailed experimental procedures for cell synchronization, cell transfection, and inhibitor treatments are in the Supporting information.

Guo X., Ramirez I., Garcia Y.A., Velasquez E.F., Gholkar A.A., Cohn W., Whitelegge J.P., Tofig B., Damoiseaux R, & Torres J.Z. (2021). DUSP7 regulates the activity of ERK2 to promote proper chromosome alignment during cell division. The Journal of Biological Chemistry, 296, 100676.

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Culturing KF1 Cells in MEM Medium

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KF1 cells were grown from stocks and maintained in Minimum Essential Media (MEM; Corning Inc., Corning, NY) supplemented with 7.5% Fetal Bovine Serum (FBS), L-glutamine, and Penicillin/Streptomycin (Genesee Scientific, San Diego, CA) at 20°C.

Quijano Cardé E.M., Yazdi Z., Yun S., Hu R., Knych H., Imai D.M, & Soto E. (2020). Pharmacokinetic and Efficacy Study of Acyclovir Against Cyprinid Herpesvirus 3 in Cyprinus carpio. Frontiers in Veterinary Science, 7, 587952.

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Lymphocyte Stimulation and Cytokine Analysis

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Single cell suspensions from aseptically removed the head and neck lymph nodes (HNLNs), mesenteric LNs (MLNs), and spleens were prepared as previously described [35 (link)]. Lymphocytes were cultured in a complete medium: RPMI 1640 with 2 mM l-glutamine (Genesee Scientific, El Cajon, CA) containing 10% fetal bovine serum (Atlanta Biologicals, Oakwood, Georgia), plus supplements (Invitrogen, Carlsbad, CA), 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids. Lymphocytes were cultured at 10⁶ cells/well in 96-well, round-bottomed tissue culture plates (Millipore, Billerica, MA) coated with 5 μg/mL anti-CD3 mAb (clone 17A2; Invitrogen, Carlsbad, CA, USA) plus 2.5 μg/mL of soluble anti-CD28 mAb (clone 37.51; Invitrogen) was stimulated for 48 h at 37 °C. Lymphocytes were stimulated in triplicate for 2 days for flow cytometry analysis or for 4 days for collection of cell culture supernatants, which were then stored at − 20 °C until assayed by cytokine-specific ELISAs.

Akgul A., Maddaloni M., Jun S.M., Nelson A.S., Odreman V.A., Hoffman C., Bhagyaraj E., Voigt A., Abbott J.R., Nguyen C.Q, & Pascual D.W. (2021). Stimulation of regulatory T cells with Lactococcus lactis expressing enterotoxigenic E. coli colonization factor antigen 1 retains salivary flow in a genetic model of Sjögren’s syndrome. Arthritis Research & Therapy, 23, 99.

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Cultured Glioma and Microglia Cells

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Mouse glioma cell line GL261-GFP.Luciferase (GL261; established and acquired from the lab of the late Dr. John Ohlfest [9 (link)]), rat glioma cell line GS-9L (9L; ECACC, 94110705), mouse microglia BV2 cell line ([10 (link)], acquired from the lab of Dr. Ling Li), and Vero cell line (African Green Monkey kidney epithelium; ATCC, CCL-81) were maintained with media changes every 48 hours and cells were passaged when reaching 80% confluence using TrypLE. Glioma media consisted of DMEM high glucose and L-glutamine (Genesee Scientific 25–500), supplemented with 10% Fetal Bovine Serum (Corning 35-011-CV), 1% Penicillin-Streptomycin (HyClone SV30010), 1% MEM NEAA (Gibco 1140–050). Vero media consisted of MEM Earle Salts supplemented with L-glutamine (Genesee Scientific 25–504), 10% Fetal Bovine Serum (Corning 35-011-CV), 1% Penicillin-Streptomycin (HyClone SV30010), 1% MEM NEAA (Gibco 1140–050).

Crane A.T., Chrostek M.R., Krishna V.D., Shiao M., Toman N.G., Pearce C.M., Tran S.K., Sipe C.J., Guo W., Voth J.P., Vaid S., Xie H., Lu W.C., Swanson W., Grande A.W., Schleiss M.R., Bierle C.J., Cheeran M.C, & Low W.C. (2020). Zika virus-based immunotherapy enhances long-term survival of rodents with brain tumors through upregulation of memory T-cells. PLoS ONE, 15(10), e0232858.

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