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Dmem powder

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DMEM powder is a cell culture medium used for the growth and maintenance of various cell types in vitro. It provides a balanced salt solution, amino acids, vitamins, and other essential nutrients required for cell proliferation and survival.

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Lab products found in correlation

Rock inhibitor y27632, by Cayman Chemical (2 mentions) Chelex 100, by Merck Group (2 mentions) Gentamycin, by Thermo Fisher Scientific (1 mentions) Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions) Sodium bicarbonate, by Merck Group (1 mentions) Fetal bovine serum (fbs), by Omega Scientific (1 mentions) 6 well plate, by Corning (1 mentions) Seakem le agarose, by Lonza (1 mentions)

11 protocols using dmem powder

1

Amino Acid Starvation and Re-Feed on Tumor Cells

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To examine the direct effects of amino acid starvation and re-feed on tumor cells, amino acid-deficient and -sufficient media were prepared. Amino acid-free (AA−) medium was prepared using Dulbecco’s Modified Eagle’s Medium (DMEM) powder (D9800-27, US Biological Life Science), d-Glucose (4.5 g/L, the same concentration as in commercially-available DMEM) and sodium bicarbonate (3.7 g/L, the same concentration as in commercially-available DMEM), and supplemented with 10% fetal bovine serum (FBS). Amino acid-sufficient (AA+) medium was prepared by adding appropriate amino acids to AA− medium to reach the same amino acid composition and content as commercially-available DMEM. Medium containing single amino acids (Gln or Leu) was prepared from AA− medium by adding individual amino acids at the same concentration as that present in AA+ medium. Media deficient for single amino acids (Gln or Leu) or a combination (Gln and Leu) thereof were prepared by adding amino acids to AA− medium, excluding the individual amino acids (with the same amino acid composition and content as in AA+ medium). Amino acids used in this study are shown in Supplementary Table 4.
Wang Z., Li B., Li S., Lin W., Wang Z., Wang S., Chen W., Shi W., Chen T., Zhou H., Yinwang E., Zhang W., Mou H., Chai X., Zhang J., Lu Z, & Ye Z. (2022). Metabolic control of CD47 expression through LAT2-mediated amino acid uptake promotes tumor immune evasion. Nature Communications, 13, 6308.
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2

Plaque Purification of CTL2M Mutant Hits

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Plaque purification of CTL2M mutant hits was performed as previously described (Kędzior and Bastidas, 2019 (link)). Confluent Vero cells in 6-well plates (Corning) were infected with 10-fold serial dilutions of C.t. seed preps. At 2 hpi, cells were covered with a DMEM-agar overlay, made from a filtered solution of DMEM powder (US Biological) and 22 μM sodium bicarbonate (Sigma) supplemented with 1% MEM non-essential amino acids (Gibco), 10% FBS (Omega Scientific), 0.025 mg/mL gentamycin (Gibco), 100 μg/mL L-Trp and 0.54% SeaKem LE agarose (Lonza). Between 10 to 20 days post-infection, individual clones were cored using a sterile p1000 tip, re-infected into a fresh monolayer of Vero cells, titered and stored in SPG buffer at −80°C.
An initial IR assay tested three to five clones for all plaque purified strains (except one hit, which yielded a single purified clone). An independent follow-up IR assay tested the two most restricted clones from the first experiment. The average of these two experiments is shown in Fig. 1D.
Walsh S.C., Reitano J.R., Dickinson M.S., Kutsch M., Hernandez D., Barnes A.B., Schott B.H., Wang L., Ko D.C., Kim S.Y., Valdivia R.H., Bastidas R.J, & Coers J. (2022). The bacterial effector GarD shields Chlamydia trachomatis inclusions from RNF213-mediated ubiquitylation and destruction. Cell host & microbe, 30(12), 1671-1684.e9.
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3

Immortalized Keratinocyte Cell Culture

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Cell Culture and Reagents: The HaCaT line of immortalized keratinocytes was provided by Yu-Ying He (University of Chicago), and was originally developed by the laboratory of Norbert Fusenig (Schoop et al., 1999) . HaCaTs were maintained in low calcium DMEM prepared from calcium-free DMEM powder (US Biological,#09800), spiked with 40 μM CaCl2, penicillinstreptomycin, L-glutamine and 10% fetal bovine serum depleted of calcium by Chelex-100 (Sigma) treatment. For calcium-induced differentiation experiments, calcium concentration was raised to 2.8 mM for the indicated time span. ROCK-inhibitor (Y-27632, Cayman Chemical) was used at 10 μM.
Harmon R.M., Devany J., & Gardel M.L. (2021). Dia1 Coordinates Differentiation and Cell Sorting in a Stratified Epithelium.
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4

Preparation of DMEM culture media

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DMEM powder (4.16g, US biological) and 1.85g sodium bicarbonate were dissolved in 400 ml H2O. To the solution, 1.75 g of glucose, 5 ml of 100x sodium pyruvate (final conc. = 1mM) and 50 ml of dialyzed fetal calf serum were added. 150 µl of 6N HCl was slowly added to bring the pH to 7.4. The final volume was adjusted to 500 ml. The media was filtered through 0.2 µm filter and kept at 4 °C.
An H., Ordureau A., Paulo J.A., Shoemaker C.J., Denic V, & Harper J.W. (2019). TEX264 is an ER-resident ATG8-interacting protein critical for endoplasmic reticulum remodeling during nutrient stress. Molecular cell, 74(5), 891-908.e10.
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5

HaCaT Keratinocyte Differentiation Induction

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The HaCaT line of immortalized keratinocytes was provided by Yu-Ying He (University of Chicago) and was originally developed by the laboratory of Norbert Fusenig (Schoop et al., 1999 (link)). HaCaTs were maintained in low calcium DMEM prepared from calcium-free DMEM powder (#09800; US Biological), spiked with 40 μM CaCl2, penicillin-streptomycin, L-glutamine, and 10% fetal bovine serum depleted of calcium by Chelex-100 (Sigma-Aldrich) treatment. For calcium-induced differentiation experiments, calcium concentration was raised to 2.8 mM for the indicated time span. ROCK-inhibitor (Y-27632; Cayman Chemical) was used at 10 μM.
Harmon R.M., Devany J, & Gardel M.L. (2022). Dia1 coordinates differentiation and cell sorting in a stratified epithelium. The Journal of Cell Biology, 221(5), e202101008.
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6

Amino Acid Withdrawal Assay Protocol

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Base-medium for the indicated amino acid withdrawal was prepared by dissolving DMEM powder (8.32g, US biological), 3.7 g/L sodium bicarbonate, and 3.5 g/L glucose, 30 mg/L glycine, 63 mg/L cystine 2HCl, 580 mg/L glutamine, 42 mg/L histidine HCl-H2O, 105 mg/L isoleucine, 66 mg/L phenylalanine, 42 mg/L serine, 95 mg/L threonine, 16 mg/L tryptophan, 104 mg/L tyrosine 2Na 2H2O, 94 mg/L valine, and 105 mg/L leucine in 880 mL H2O, and pH titrated to pH 7.2 using 2M HCl. The base-media was filtered through 0.2 μm filter and kept at 4 °C. Before each experiment, 0.9 equivalence (v/v) of the base-medium was added by 0.1 equivalence (v/v) of dialyzed bovine serum albumin, 0.01 equivalence (v/v) of 100 × sodium pyruvate (final conc. 1mM) and 0.001 equivalence of 1000 × amino acids (methionine stock: 37.5 mg/ml (final 37.5 mg/L = 250 μM), Lysine HCl stock: 146 mg/ml (final 146 mg/L), Arginine HCl stock: 84 mg/ml (final 84 mg/L) except the limiting amino acid.
An H., Ordureau A., Schultz M., Paulo J.A, & Harper J.W. (2020). Systematic Quantitative Analysis of Ribosome Inventory Upon Nutrient Stress. Nature, 583(7815), 303-309.
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7

Preparation of DMEM Growth Media

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DMEM powder (4.16 g, US biological) and 1.85 g sodium bicarbonate were dissolved in 400 ml H2O. To the solution, 1.75 g of glucose, 5 ml of 100x sodium pyruvate (final conc. = 1 mM) and 50 ml of dialyzed fetal calf serum were added. 150 μl of 6N HCl was slowly added to bring the pH to 7.4. The final volume was adjusted to 500 ml. The media was filtered through 0.2 μm filter and kept at 4 °C.
An H., Ordureau A., Schultz M., Paulo J.A, & Harper J.W. (2020). Systematic Quantitative Analysis of Ribosome Inventory Upon Nutrient Stress. Nature, 583(7815), 303-309.
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8

Leucine-Free Cell Culture Medium

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Solubility of Leucine in H2O was too low to be added at the last step as a concentrate. Therefore, leucine free medium had to be prepared separately from the -Lys and -Arg medium. DMEM powder (8.32g, US biological), 3.7g/L sodium bicarbonate, and 3.5 g/L glucose, 30 mg/L glycine, 63 mg/L cystine 2HCl, 580 mg/L glutamine, 42 mg/L histidine HCl-H2O, 105 mg/L isoleucine, 66 mg/L phenylalanine, 42 mg/L serine, 95 mg/L threonine, 16 mg/L tryptophan, 104 mg/L tyrosine 2Na 2H2O, 94 mg/L valine, 146 mg/L Lys, and 84 mg/L Arg were dissolved in 880 mL H2O, and pH titrated to pH7.2 using 2M HCl. The base-media was filtered through 0.2 micron filter and kept at 4 °C. Before each experiment, 0.9 equivalence (v/v) of the base medium was added by 0.1 equivalence (v/v) of dialyzed bovine serum albumin, 0.01 equivalence of 100X sodium pyruvate (v/v), and 0.001 equivalence (v/v) of 1000X methionine (final 37.5 mg/L = 250 μM) or 0.01 equivalence (v/v) of 100X AHA (final 250 μM).
An H., Ordureau A., Schultz M., Paulo J.A, & Harper J.W. (2020). Systematic Quantitative Analysis of Ribosome Inventory Upon Nutrient Stress. Nature, 583(7815), 303-309.
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9

Leucine-Free Cell Culture Medium

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Solubility of Leucine in H2O was too low to be added at the last step as a concentrate. Therefore, leucine free medium had to be prepared separately from the -Lys and -Arg medium. DMEM powder (8.32g, US biological), 3.7g/L sodium bicarbonate, and 3.5 g/L glucose, 30 mg/L glycine, 63 mg/L cystine 2HCl, 580 mg/L glutamine, 42 mg/L histidine HCl-H2O, 105 mg/L isoleucine, 66 mg/L phenylalanine, 42 mg/L serine, 95 mg/L threonine, 16 mg/L tryptophan, 104 mg/L tyrosine 2Na 2H2O, 94 mg/L valine, 146 mg/L Lys, and 84 mg/L Arg were dissolved in 880 mL H2O, and pH titrated to pH7.2 using 2M HCl. The base-media was filtered through 0.2 micron filter and kept at 4 °C. Before each experiment, 0.9 equivalence (v/v) of the base medium was added by 0.1 equivalence (v/v) of dialyzed bovine serum albumin, 0.01 equivalence of 100X sodium pyruvate (v/v), and 0.001 equivalence (v/v) of 1000X methionine (final 37.5 mg/L = 250 μM) or 0.01 equivalence (v/v) of 100X AHA (final 250 μM).
An H., Ordureau A., Schultz M., Paulo J.A, & Harper J.W. (2020). Systematic Quantitative Analysis of Ribosome Inventory Upon Nutrient Stress. Nature, 583(7815), 303-309.
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10

Amino Acid Withdrawal Assay Protocol

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Base-medium for the indicated amino acid withdrawal was prepared by dissolving DMEM powder (8.32g, US biological), 3.7 g/L sodium bicarbonate, and 3.5 g/L glucose, 30 mg/L glycine, 63 mg/L cystine 2HCl, 580 mg/L glutamine, 42 mg/L histidine HCl-H2O, 105 mg/L isoleucine, 66 mg/L phenylalanine, 42 mg/L serine, 95 mg/L threonine, 16 mg/L tryptophan, 104 mg/L tyrosine 2Na 2H2O, 94 mg/L valine, and 105 mg/L leucine in 880 mL H2O, and pH titrated to pH 7.2 using 2M HCl. The base-media was filtered through 0.2 μm filter and kept at 4 °C. Before each experiment, 0.9 equivalence (v/v) of the base-medium was added by 0.1 equivalence (v/v) of dialyzed bovine serum albumin, 0.01 equivalence (v/v) of 100 × sodium pyruvate (final conc. 1mM) and 0.001 equivalence of 1000 × amino acids (methionine stock: 37.5 mg/ml (final 37.5 mg/L = 250 μM), Lysine HCl stock: 146 mg/ml (final 146 mg/L), Arginine HCl stock: 84 mg/ml (final 84 mg/L) except the limiting amino acid.
An H., Ordureau A., Schultz M., Paulo J.A, & Harper J.W. (2020). Systematic Quantitative Analysis of Ribosome Inventory Upon Nutrient Stress. Nature, 583(7815), 303-309.
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