HepG2 cells (ATCC) for use in cytotoxicity assays (HepG2/Gal) were maintained for three passages in glucose-free RPMI 1640 media (VWR International, Suwanee, GA) supplemented with 7.5 mM galactose (Sigma-Aldrich), 10% fetal bovine serum (FBS; Atlanta Biological, Flowery Branch, GA), 2 mM l -glutamine (VWR), and 1× penicillin-streptomycin (VWR). HepG2 cells for use in other assays were maintained in glucose-containing RPMI 1640 (VWR) supplemented with FBS, 2 mM l -glutamine, and 1× penicillin-streptomycin. CEM cells (ATCC) were maintained in RPMI 1640 media supplemented with 10% FBS, 2 mM l -glutamine and 1× penicillin-streptomycin. PC-3 cells (ATCC CRL-1435) were incubated in Ham’s F-12K complete medium (Thermo Fisher Scientific, Waltham, MA) containing 10% FBS HI, 2 mM l -glutamine, and 1× penicillin-streptomycin.
L glutamine
Manufactured by Avantor
Sourced in United States, Sweden, China
L-glutamine is a non-essential amino acid that plays a crucial role in various metabolic processes in the body. It is a key component in the production of proteins, and it also serves as a fuel source for certain cells, particularly those in the gastrointestinal tract and the immune system.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Avantor (21 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (16 mentions)
Penicillin, by Avantor (14 mentions)
Streptomycin, by Avantor (12 mentions)
Penicillin streptomycin, by Avantor (10 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions)
Rpmi 1640, by Avantor (6 mentions)
Hepes, by Avantor (6 mentions)
Non essential amino acids (neaa), by Avantor (6 mentions)
Penicillin, by Thermo Fisher Scientific (6 mentions)
Rpmi 1640, by Thermo Fisher Scientific (5 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (5 mentions)
Sodium pyruvate, by Avantor (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
β mercaptoethanol, by Merck Group (5 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Percoll, by GE Healthcare (4 mentions)
Phosphate buffered saline (pbs), by Avantor (4 mentions)
Insulin, by Merck Group (4 mentions)
Transferrin, by Merck Group (4 mentions)
72 protocols using l glutamine
1
Optimized Cell Culture Conditions
2
Experimental Protocol for Mouse EAE Model
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The reagents used in this study were purchased as described: THC and CBD from Cayman Chemical (Michigan, USA), myelin oligodendrocyte glycoprotein (MOG35−55) peptide H-MEVGWYRSPFSRVVHLYRNGK-OH (PolyPeptide Laboratories, San Diego, CA, USA). Mycobacterium tuberculosis (strain H37Ra) (BD, Franklin Lakes, NJ, USA), complete Freund's adjuvant (Fisher, Hampton, NH, USA), Pertussis toxin (List Biological Laboratories, Campbell, CA, USA), Percoll, GE Healthcare Life Sciences (Pittsburgh, PA, USA); Neural Tissue Dissociation Kit (P) (Miltenyi Biotech, Auburn, CA, USA), RBC lysis buffer (Sigma-Aldrich, St. Louis, MO, USA), RPMI 1640, l-glutamine, HEPES, phosphate-buffered saline, and fetal bovine serum (VWR, West Chester, PA, USA), ELISA Max Kits IL-10, IL-17A, IFN-γ, IL-6, IL-1β, TNF-α, and TGF-β and FITC Annexin V/-PI apoptosis kit (Biolegend, San Diego, CA). EasySep PE selection kit (Stemcell Technologies, Cambridge, MA, USA), Propidium Iodide (PI)/RNase Staining Solution (Cell Signaling Technology, Danvers, MA, USA), miRNeasy Mini Kit, miScript II RT Kit and miRNAs primers (Qiagen, Valencia, CA), mRNAs primers (Integrated DNA technologies, Coralville, IA, USA) and SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA).
3
Cultivation and Maintenance of Cell Lines for VEEV Study
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Celecoxib (S1261), Rolipram (S2127), and Tofacitinib (S5001) were obtained from SelleckChem. The live-attenuated TC-83 vaccine strain of VEEV was acquired from BEI Resources. HMC3 human microglial cells (ATCC CRL-3304), U87 MG human brain astrocytoma cells (ATCC HTB-14), and VERO African green monkey kidney cells (ATCC CCL-81) were obtained from the American Type Culture Collection. U87 MG cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (VWR Life Science VWRL0102-500) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco 10,437,028), 1% Penicillin/Streptomycin (Gibco 15-140-122), and 1% L-Glutamine (VWR Life Science VWRL0131-0100) at 37 °C and 5% CO2. HMC3 cells were maintained in Eagle’s minimal essential medium (EMEM) (VWR Life Sciences 10128-214) supplemented with 10% FBS and 1% Penicillin/Streptomycin at 37 °C and 5% CO2. African green monkey kidney cells (VEROs) were maintained in DMEM supplemented with 5% heat-inactivated fetal bovine essence (FBE) (VWR Life Sciences 10803-034), 1% Penicillin/Streptomycin, and 1% L-Glutamine at 37 °C and 5% CO2. All reagents for cell maintenance were pre-warmed to 37 °C before use.
4
Culturing JIMT-1 Breast Cancer and Human Dermal Fibroblasts
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The human breast carcinoma cell line JIMT-1 was purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). The cells were tested for mycoplasma during the experimental period and were found to be negative (Eurofins Scientific, Cologne, Germany). HDFs were purchased from Sigma-Aldrich Sweden AB (Stockholm, Sweden). The HDFs were maximally used for up to 7 passages. The cell lines were cultured in Dulbecco’s modified Eagle medium/Ham’s F-12 (1:1) supplemented with 5 % heat-inactivated donor herd horse serum (DHHS) (Sigma-Aldrich Sweden AB), 5 μg/mL insulin (Sigma-Aldrich Sweden AB), 10 ng/mL epidermal growth factor (Lonza, Basel, Switzerland), 0.5 μg/mL hydrocortisone (Sigma-Aldrich Sweden AB), 1 mM Na-pyruvate (Sigma-Aldrich Sweden AB), 50 μg/mL transferrin (Sigma-Aldrich Sweden AB), 2 mM l -glutamine (VWR, Lund, Sweden), 1 mM non-essential amino acids (VWR), 100 μg/mL streptomycin (VWR), and 100 U/mL penicillin (VWR). The cell lines were maintained in a humidified incubator (95 % humidity) with 5 % CO2 in air at 37 °C (CO2 incubator). The cells were passaged twice a week using Accutase™ (Sigma-Aldrich Sweden AB).
5
Culturing and Maintaining JIMT-1 and HDF Cell Lines
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The human breast carcinoma cell line JIMT-1 (ACC-589) was purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). The cells were tested for mycoplasma during the experimental period and were found to be negative (Eurofins Scientific, Cologne, Germany). HDFs (106-05a) were purchased from Sigma-Aldrich Sweden AB (Stockholm, Sweden). The HDFs were maximally used between passages 3–6. The cell lines were cultured in Dulbecco’s modified Eagle medium/Ham´s F-12 (1:1) supplemented with 5% heat-inactivated donor herd horse serum (DHHS) (Sigma-Aldrich Sweden AB), 5 μg/ml insulin (Sigma-Aldrich Sweden AB), 10 ng/ml epidermal growth factor (Lonza, Basel, Switzerland), 0.5 µg/ml hydrocortisone (Sigma-Aldrich Sweden AB), 1 mM Na-pyruvate (Sigma-Aldrich Sweden AB), 50 µg/ml transferrin (Sigma-Aldrich Sweden AB), 2 mM L-glutamine (VWR, Lund, Sweden), 1 mM non-essential amino acids (VWR), 100 μg/ml streptomycin (VWR), and 100 U/ml penicillin (VWR). The cell lines were maintained in a humidified incubator (95% humidity) with 5% CO2 in air at 37 ºC (CO2 incubator). The cells were passaged twice a week using Accutase (Sigma-Aldrich Sweden AB).
6
Cytokine Production in Stimulated PBMCs
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PBMC were prepared using Ficoll (GE Healthcare, Mississauga, Canada) and cultured (triplicates, 350,000 cells/round bottom well in 200 μL, 24 h) in medium alone or with stimuli. Medium consisted of RPMI-1640 (Thermo Fisher Scientific, Mississauga, Canada) supplemented with 10% fetal bovine serum (GE Healthcare, Mississauga, Canada), 1% l -glutamine (VWR International, Mississauga, Canada), 1% Antibiotic–Antimycotic (Thermo Fisher Scientific), and 0.1% 2-mercaptoethanol (Thermo Fisher Scientific). Innate stimuli used included TLR4 ligand LPS (0.4 ng/mL, InvivoGen, San Diego, CA) or RLR ligand Poly(I:C)/Lyovec (250 ng/mL, InvivoGen). All PBMC samples were cultured the day they were drawn. Supernatants were aliquoted and examined in parallel without ever freezing and after five F/T cycles at −80 and 37 °C.
7
Cell Line Culture and Authentication
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MDA-MB-468, MDA-MB-231, MCF7, T47D, BT474 and ZR-75-1 cells were from the American Type Culture Collection (Manassas, VA); all other cell lines were a gift from Dr. Norma O’Donovan (Dublin City University, Dublin, Ireland). MCF7 cells were maintained in DMEM-high glucose media (ThermoFisher Scientific, Waltham, MA) with 10% fetal bovine serum (FBS) (Atlanta Biologics, Flowery Branch, GA), 1% penicillin-streptomycin and 2 mM L-glutamine (VWR, Radnor, PA), 1 mM sodium pyruvate (Sigma-Aldrich, St Louis, MO) and 5 mg insulin (Sigma-Aldrich). MDA-MB-468, MDA-MB-231, SKBR3 and JIMT-1 cells were maintained in DMEM-high glucose media (ThermoFisher Scientific) with 10% FBS, 1% penicillin-streptomycin and 2 mM L-glutamine. All other cell lines were maintained in RPMI-1640 (ThermoFisher Scientific) with 10% FBS, 1% penicillin-streptomycin and 2mM L-glutamine. All cell lines were routinely confirmed to be free of Mycoplasma (MycoAlert PLUS mycoplasma detection kit (Lonza, Alpharetta, GA)) and were authenticated by STR profiling by the University of Arizona Genetic Core (Tuscon, AZ) or by Source Bioscience (Santa Fe Springs, CA) in 2016. THZ1 was purchased from MedChem Express (Monmouth Junction, NJ) as a 10 mM stock solution in DMSO. Erlotinib was purchased from LC Laboratories (Woburn, MA).
8
Culturing Embryonic Stem Cells and HEK293T
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CyT49 embryonic stem cells were maintained in DMEM F12 (without L-glutamine; VWR) + 10% knockout serum replacement (KSR; Thermo Fisher Scientific), 1% non-essential amino acids (NEAA; Thermo Fisher Scientific), 1% GlutaMAX (Thermo Fisher Scientific), 0.2% β-mercaptoethanol (Thermo Fisher Scientific) and 1% penicillin–streptomycin (Thermo Fisher Scientific). HEK293T were maintained in DMEM F12 containing 100 units/mL penicillin and 100 mg/mL streptomycin sulfate supplemented with 10% foetal bovine serum (FBS).
9
Maintenance of Toxoplasma Gondii Strains
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All parasite strains, including parental RHΔhxgprt, TgSKP1SF [20 (link)], TATiΔku80 [67 (link)] and RHΔhxgprtΔku80) strains [68 (link)], were maintained in human foreskin fibroblasts (ATCC; Manassas, VA) in Dulbecco’s Modification of Eagle’s Medium (DMEM) (VWR; Radnor, PA) supplemented with 10% fetal bovine serum (VWR), 2 mM L-glutamine (VWR), and 100 IU/ml penicillin– 100 μg/ml streptomycin (VWR). Parasites were released from host cells by passage through a 27-gauge needle [69 (link)]. All parasite strains and host cell lines were routinely tested for Mycoplasma contamination with the MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland) and found to be negative.
10
Culturing and SILAC-labeling of Cell Lines
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HeLa, 293T, and Huh7 cells were maintained in Dulbecco’s Modified Eagle’s Medium (D6428, Sigma) supplemented with 10% FBS (35-010-CV, Corning) at 37 °C, 5% CO2. 832/13 cells were maintained in RPMI1640 (11875-093, Invitrogen) supplemented with 2 mM l -glutamine (25030-081, Invitrogen), 1 mM Na-pyruvate (11360-070, Invitrogen), 10 mM HEPES (15630-080, Invitrogen), 0.05 mM 2-mercaptoethanol (M722, Sigma), and 10% FBS at 37 °C, 5% CO2. SILAC-labeling was done using the Pierce SILAC-protein quantitation kit (1863108, Thermo), supplemented with 2 mM l -glutamine (02-0131-0200, VWR), and 10 µg/mL l -proline (88211, Thermo). Cells were passaged at least five times in their respective SILAC-labeled media (>10 doublings). For the comparative LEAP-RBP experiment, HeLa cells were maintained as described above and treated with DMSO (negative control) or 2 µg/mL Harringtonine (15361, Cayman Chemical Company) for 30 min at 37 °C, 5% CO2; Harringtonine (HT) was prepared as a 1000× stock in DMSO.
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