MRC5 human lung fibroblasts (ATCC #CCL-171) were maintained in DMEM with 4.5 g/L glucose (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) in a humidified incubator at 37°C with 5% CO2. Cells were cultured for five passages in SILAC medium supplemented with 10% dialyzed FBS to obtain near-uniform (>99%) labeling in accordance with published methods [30 (link)]. SILAC medium was prepared by dissolving the components of DMEM except for lysine and arginine, and then supplementing with either “light” isotope lysine and arginine (Sigma-Aldrich) or uniformly 13C and 15N-labeled lysine (U-13C6, 15N2; Δ8 Da) and arginine (U-13C6, 15N4; Δ10 Da) (Cambridge Isotope Laboratories) for the “heavy” labeling condition. Light- and heavy-SILAC labeled cells were infected with pUL97-expressing or pUL97-deficient viruses. The HCMV strain AD169 derivatives, UL97-K355Q and its revertant UL97-Q355K, were the generous gift of Dr. Donald Coen (Harvard Medical School), and have been previously described [16 (link)]. Stocks of HCMV were grown in MRC5 cells in roller bottles and viral titers were determined by 50% tissue culture infectious dose (TCID50) assay.
Dmem with 4.5 g l glucose
Manufactured by Merck Group
Sourced in United States
DMEM with 4.5 g/L glucose is a cell culture medium formulation. It contains 4.5 grams of glucose per liter. This medium is designed to support the growth and maintenance of various cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Human serum, by Gemini Bio (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Dialyzed fbs, by Merck Group (1 mentions)
Hek293a cells, by Thermo Fisher Scientific (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
G418 disulfate, by Caisson (1 mentions)
5 protocols using dmem with 4.5 g l glucose
1
SILAC-Based Quantitative HCMV Proteomics
2
Maintenance and Differentiation of HEK293T, TZM-bl, and Human Monocyte-Derived Macrophages
3
Maintenance and Differentiation of HEK293T, TZM-bl, and Human Monocyte-Derived Macrophages
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HEK293T and TZM-bl cells were maintained in DMEM with 4.5 g/L glucose (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin in a 37°C and 5.0% CO2 environment. Human monocytes from multiple anonymous normal donors were elutriated as described previously (Gruber et al., 1995 (link)). Elutriated monocytes were obtained from the NIH blood bank under protocol 99-CC-0168: “Collection and Distribution of Blood Components from Healthy Donors for In Vitro Research Use.” Monocytes (2 × 106 cells/well in a 12-well plate) were cultured in 1 mL of complete DMEM supplemented with 10% pooled human serum (Gemini Bio-Products, West Sacramento, CA, USA) for 5–7 days to allow differentiation into macrophages (MDMs).
4
Culturing of Human Cell Lines
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HEK293T and TZM-bl cells were maintained in DMEM with 4.5 g/L glucose (Sigma-Aldrich, St. Louis, MO) that was supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. THP-1 cells were grown in complete RPMI medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The differentiation of THP-1 cells was achieved by growing cells for 48 h in the presence of PMA (100 ng/mL), and this was followed by a resting period of 24 h, as described (42 (link)). Elutriated human monocytes from multiple anonymous normal donors were obtained from the NIH blood bank under protocol 99-CC-0168: “Collection and Distribution of Blood Components from Healthy Donors for In Vitro Research Use”. Monocytes (4 × 106 cells/well in a 6-well plate) were cultured in 2 mL of complete DMEM supplemented with 10% pooled human serum (Gemini Bio-Products, West Sacramento, CA) and 1 mM sodium pyruvate (Thermo Fisher Scientific, Atlanta, GA 30384) for 5 to 7 days to allow for differentiation into macrophages (MDMs).
5
Cell Line Characterization for Nutrient Signaling
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HELA, HEK293T, U2OS, mouse embryonic fibroblasts (MEF) cells deficient in GCN2 and their littermate-derived wild-type control cells were obtained from ATCC. HEK293A cells were purchased from Thermo Fisher Scientific. RagA/B CRISPR/Cas9 knockout cells and control cells were kindly provided by Drs. Kun-Liang Guan and Jenna L. Jewell (University of California San Diego46 (link)). Mouse embryonic fibroblasts deficient in p14/LAMTOR2 and wild-type control cells were kindly provided by Drs. David Sabatini (Whitehead Institute) and Brendan Manning (Harvard School of Public Health). U2OS cells stably expressing mCherry-LAMP1 were generated through transfection of pLAMP1-mCherry and selection was carried out with 2 mg/mL G418 disulfate (Caisson Lab) whereafter a single colony of expressing cells was isolated.
All cell lines were cultured in DMEM with 4.5 g/L glucose (Life Technologies) with 10% FBS (Sigma-Aldrich) and 1% penicillin–streptomycin (Thermo Fisher Scientific). For amino acid starvation experiments, DMEM with 4.5 g/L glucose containing all amino acids (control) or no amino acids (-AA) were purchased from the Memorial Sloan Kettering Cancer Center Media Core Facility (New York, NY) and combined with dialyzed FBS (Sigma-Aldrich).
All cell lines were cultured in DMEM with 4.5 g/L glucose (Life Technologies) with 10% FBS (Sigma-Aldrich) and 1% penicillin–streptomycin (Thermo Fisher Scientific). For amino acid starvation experiments, DMEM with 4.5 g/L glucose containing all amino acids (control) or no amino acids (-AA) were purchased from the Memorial Sloan Kettering Cancer Center Media Core Facility (New York, NY) and combined with dialyzed FBS (Sigma-Aldrich).
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