MEM media was purchased from Life Technologies (Grand Island, NY). G418 Disulfate was obtained from Caisson Labs (G030-5GM). Fura-2AM was purchased from Molecular Probes (Eugene, OR). DNP25-BSA was from ThermoFisher Scientific (Waltham, MA; catalogue #A23018), custom DF3 peptide synthesized by and purchased from Anaspec (Fremont, CA) and anti-DNP-IgE was affinity-purified from ascites (Covance, Denver, PA) according to the methods of Liu et al (28 ). AlexaFluor647- (Thermofisher Scientific #A-20006), Janelia Fluor 646- (Tocris #6148) and CF640R- (Biotium #92108) labeled IgE lots were prepared using NHS Ester conjugation. Antibodies used are listed as follows: anti-pY PY20/99 cocktail (SantaCruz; sc-508, sc-7020), anti-rabbit-HRP secondary (SantaCruz; sc-2004, Cell Signaling #7074), anti-FcεRIγ (EMD Millipore; 06–727), Goat anti-rat IgE (Abnova; PAB29749). Poly-L lysine was purchased from Sigma-Aldrich (P1524-100MG), 18:1 (Δ9-Cis) PC (DOPC) and 16:0 DNP Cap PE lipids were purchased from Avanti Polar Lipids (850375C and 810528C, respectively). DNP-Peg12-NHS ester was purchased from BroadPharm (BP-22397). All live-cell RBL imaging was performed in modified Hank’s buffered salt solution (additional 10 mM Hepes, 0.05% w/v BSA, 5.45 mM glucose, 0.88 mM MgSO4, 1.79 mM CaCl2, 16.67 mM NaHCO3).
G418 disulfate
Manufactured by Caisson
G418 Disulfate is a broad-spectrum antibiotic used for selection of eukaryotic cells that have been transfected with the neomycin resistance gene. It inhibits protein synthesis in eukaryotic cells by binding to the 80S ribosomal subunit.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Mem media, by Thermo Fisher Scientific (1 mentions)
Dnp25 bsa, by Thermo Fisher Scientific (1 mentions)
Janelia fluor 646, by Bio-Techne (1 mentions)
Cf640r, by Biotium (1 mentions)
Anti dnp ige, by Fortrea (1 mentions)
18 1 δ9 cis pc dopc, by Avanti Polar Lipids (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Hek293a cells, by Thermo Fisher Scientific (1 mentions)
Dialyzed fbs, by Merck Group (1 mentions)
Dmem with 4.5 g l glucose, by Merck Group (1 mentions)
2 protocols using g418 disulfate
1
Mast Cell Degranulation Assay
2
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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