For lysosomal degradation inhibition experiments, cells were treated with 50 µM chloroquine for 6 h; to inhibit mTORC1 activation, cells were treated with AZD8055, a mTORC1 inhibitor, for 25 h. To induce mTORC1 activation by essential amino acid, cells were starved in amino acid-free DMEM medium (D9800-13; USBiological) for 2 h before stimulated by MEM essential amino acid solution (final concentration: 2×; 11130051; Thermo Fisher Scientific) for 30 min.
Dmem medium
Manufactured by US Biological
Sourced in United States
DMEM medium is a commonly used cell culture medium that provides essential nutrients for the growth and maintenance of various cell types. It is a balanced salt solution that includes amino acids, vitamins, and other components necessary for cell proliferation and survival.
Automatically generated - may contain errors
Lab products found in correlation
D9800 13, by US Biological (1 mentions)
Mem essential amino acid solution, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by GE Healthcare (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
Pantothenate, by Merck Group (1 mentions)
Glutamine, by US Biological (1 mentions)
Pyruvate, by Merck Group (1 mentions)
Glucose, by Thermo Fisher Scientific (1 mentions)
Pantothenate, by US Biological (1 mentions)
Epoch plate reader, by Agilent Technologies (1 mentions)
Sulforhodamine b, by Merck Group (1 mentions)
24 well plate, by Greiner (1 mentions)
3 protocols using dmem medium
1
Lysosomal Degradation and mTORC1 Modulation
2
In vitro Skeletal Muscle Cell Culture
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In vitro analysis was carried out using the murine skeletal muscle cell line C2C12. Cells were cultured, sub-cultured and differentiated (up to 7 d) in DMEM medium as previously described [18 ,24 ]. Prior to treatment with media conditioned by human serum, fully differentiated myotubes were nutrient deprived in an AA and serum free DMEM medium (US biological, Salem, MA, USA), supplemented with 1 mM sodium pyruvate (GE Healthcare, Thermo-Fisher), 1% (v/v) penicillin/streptomycin solution, 1 mM L-glutamine, 6 mM D-glucose (Sigma-Aldrich), and 34 mM NaCl (Sigma-Aldrich) (pH 7.3).
3
HMEC-1 Cell Growth Assay under Hypoxia
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HMEC-1 cells or HMEC-1 cells overexpressing xCT or SLC1A3 were seeded into 24-well plates (Greiner Bio-One) at a density of 6.5 x 10 3 cells per well in 0.5 mL medium. The following day, medium was changed to 1 mL HMEC-1 growth medium with any indicated supplements. For assays with added aspartate, medium was changed to custom DMEM medium without glucose, glutamine, or pantothenate (US Biological) supplemented with 5.55 mM glucose (Gibco), 1 mM pyruvate (Sigma), and 25 µM pantothenate (Sigma) along with supplements above: EGF, hydrocortisone, glutamine, HI-FBS. Media was buffered with sodium hydroxide to pH 7.95.
Designated plates were placed in hypoxia chamber at 1% oxygen. Cell number was measured at the indicated time points using sulforhodamine B (Sigma) staining, as previously described (42) .
Absorbance at 510 nm was read on an Epoch plate reader (BioTek). Relative growth was determined as (treatment absorbance / control absorbance).
Designated plates were placed in hypoxia chamber at 1% oxygen. Cell number was measured at the indicated time points using sulforhodamine B (Sigma) staining, as previously described (42) .
Absorbance at 510 nm was read on an Epoch plate reader (BioTek). Relative growth was determined as (treatment absorbance / control absorbance).
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