Bathocuproinedisulfonic acid bcs

Manufactured by Merck Group

Sourced in United States

Bathocuproinedisulfonic acid (BCS) is a chemical compound used in laboratory settings. It functions as a chelating agent, capable of forming stable complexes with copper ions. BCS is commonly utilized in analytical and biochemical applications that require the detection or measurement of copper.

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5 protocols using bathocuproinedisulfonic acid bcs

Culturing Mouse and Human Lymphoma Cells

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Eμ-Myc mouse lymphoma cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco), 50 μM β-mercaptoethanol (Sigma-Aldrich), 100 μM asparagine (Sigma-Aldrich), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco) at 37°C and 10% CO_2. OP9 cells were cultured in αMEM (Gibco) supplemented with 20% heat-inactivated FBS, 1 mM glutamine (Gibco), 10 mM Hepes (Gibco), 1 mM sodium pyruvate (Gibco), 50 μM β-mercaptoethanol, 100 U/mL penicillin, and 100 μg/mL streptomycin. Human cell lines were cultured in a humidified incubator at 37°C and 5% CO₂. Virus-producing 293T cells were maintained in DMEM supplemented with 10% FBS. Approximately 6 h prior to transfection, 293T cells were cultured in DME glutamax (Gibco) supplemented with 10% FBS and 25 mM Hepes. Rael-BL, Ramos-BL, Sav-BL, and BL-31 human Burkitt lymphoma-derived cell lines were cultured in RPMI 1640 supplemented with 10% FBS, 1 mM glutamine (Gibco), 1 mM sodium pyruvate (Gibco), 50 μM α-thioglycerol (Sigma-Aldrich), and 20 nM bathocuproine disulfonic acid (BCS) (Sigma-Aldrich). X50-7 and Awia lymphoblastoid cell lines were cultured in RPMI 1640 supplemented with 10% FBS and 1 mM glutamine.

Kelly G.L., Grabow S., Glaser S.P., Fitzsimmons L., Aubrey B.J., Okamoto T., Valente L.J., Robati M., Tai L., Fairlie W.D., Lee E.F., Lindstrom M.S., Wiman K.G., Huang D.C., Bouillet P., Rowe M., Rickinson A.B., Herold M.J, & Strasser A. (2014). Targeting of MCL-1 kills MYC-driven mouse and human lymphomas even when they bear mutations in p53. Genes & Development, 28(1), 58-70.

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Preparation and Treatment of Cancer Cells

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20 mM disulfiram (DSF, Sigma) stock was dissolved in DMSO and further diluted to 20 μM in PBS. 10 mM CuSO₄ (Sigma) stock was dissolved in water. 3–5 mM GC4419 (Galera Therapeutics) stock was dissolved in bicarbonate buffer. 100 mM buthionine sulfoximine (BSO, Sigma) stock was dissolved in PBS. 1 mg/mL auranofin (AUR, Enzo Life Science) stock was dissolved in ethanol and then further diluted 1:10 in PBS. 1 mg/mL cisplatin (Fresenius Kabi) stock was diluted 1:10 in PBS. 1 mg/mL doxycycline (Fisher) stock was dissolved in water. 10 mM bathocuproinedisulfonic acid (BCS, Sigma) stock was dissolved in water. For drug treatments, cells were plated at least 48 h prior to treatment and allowed to grow to 40–80% confluence. Cancer cell drug treatments were given in RPMI media with 10% FBS. Tumor vs. normal cell drug treatments were given in HBEC media. Final drug concentrations are reported in figure legends. Radiation in vitro was delivered using a 6,000 Ci ¹³⁷Cs source (JL Shepherd and Associates, San Fernando, CA) with doses of 0–6 Gy (dose rate, ≈ 0.65 Gy/min).

Falls-Hubert K.C., Butler A.L., Gui K., Anderson M., Li M., Stolwijk J.M., Rodman SN I.I.I., Solst S.R., Tomanek-Chalkley A., Searby C.C., Sheffield V.C., Sandfort V., Schmidt H., McCormick M., Wels B.R., Allen B.G., Buettner G.R., Schultz M.K, & Spitz D.R. (2020). Disulfiram causes selective hypoxic cancer cell toxicity and radio-chemo-sensitization via redox cycling of copper. Free radical biology & medicine, 150, 1-11.

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Paracoccidioides lutzii Adhesion Assay

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The Paracoccidioides lutzii 01 strain (ATCC MYA-826) was used in all experiments. The yeast phase was maintained in vitro in Fava-Netto’s medium [56 ] for 7 days at 36°C. For the adhesion assay, the fungus was incubated under three conditions: (1) complete McVeigh-Morton (MVM) medium [57 (link),58 (link)] containing glucose 1%, KH₂PO₄ 11 mM, MgSO₄.7H₂0 2 mM, CaCl₂.2H₂0 1 mM, (NH₄)₂SO₄ 15 mM, L-Asparagine 0.02%, L-Cystine 0.002%, vitamin supplements 1%, and trace element supplement 0.1%, (2) MVM without Cu reagent (MVM-W-Cu) and (3) MVM without Fe reagent. Moreover, bathocuproinedisulfonic acid (BCS) (Sigma-Aldrich, St. Louis, MO, USA) and bathophenanthroline disulfate (BPS) (Sigma-Aldrich, St. Louis, MO, USA) were used to chelate the Cu and Fe ions, respectively. The fungus maintained in Fava-Netto’s medium was transferred to the three conditions described above and incubated for 3 h at 37°C.

de Oliveira H.C., da Silva J.D., Matsumoto M.T., Marcos C.M., Peres da Silva R., Moraes da Silva R.A., Labate M.T., Labate C.A., Fusco Almeida A.M, & Mendes Giannini M.J. (2014). Alterations of protein expression in conditions of copper-deprivation for Paracoccidioides lutzii in the presence of extracellular matrix components. BMC Microbiology, 14, 302.

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Yeast Growth Under Low Copper/Iron

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For growth under low copper conditions, yeast strains were grown in low copper complete minimal media. To attain low copper conditions, the media contained yeast nitrogen base without copper and iron (YNB-CuSO₄-FeCl₃) and 100 μM Bathocuproinedisulfonic acid (BCS; Sigma-Aldrich). To achieve low iron conditions, the media was prepared as described above for low copper replacing 100 μM BCS with 100 μM Bathophenanthrolinedisulfonic acid (BPS; Sigma-Aldrich). Glassware used in these experiments was soaked in 10% nitric acid overnight to remove trace amounts of copper and iron. All yeast cells used for low copper and iron were initially grown to saturation in complete minimal media then sub-cultured into copper or iron deficient media in acid washed glassware.

Wong A., Lam E.M., Pai C., Gunderson A., Carter T.E, & Kebaara B.W. (2021). Variation of the response to metal ions and nonsense-mediated mRNA decay across different Saccharomyces cerevisiae genetic backgrounds. Yeast (Chichester, England), 38(9), 507-520.

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Copper and Iron Effects on mRNA

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For analysis of the mRNAs under low copper conditions, wild-type and NMD mutant strains were grown in low copper complete minimal (CM) media. This media contained yeast nitrogen base without copper (YNB-CuSO₄-FeCl₃) and 100 uM of bathocuproinedisulfonic acid (BCS) (Sigma-Aldrich, St. Louis, MO, USA). For analysis of the mRNAs under low iron conditions, wild-type and NMD mutant strains were grown in low iron CM media containing 100μM Bathophenanthrolinedisulfonic acid (Sigma-Aldrich). Glassware used in these experiments was soaked in 10% nitric acid overnight to remove trace amounts of copper and iron. All yeast cells used for low copper and low iron northerns were initially grown to saturation in CM media and then sub-cultured into copper or iron deficient media in acid washed glassware.
To analyze the mRNAs under high copper conditions, Wild-type and NMD mutant yeast cells were grown in CM media supplemented with 100μM copper (high copper media). As with the low copper and iron conditions, the yeast cells were first grown to saturation in CM media then sub-cultured into media supplemented with 100μM copper.

Peccarelli M., Scott T.D, & Kebaara B.W. (2019). Nonsense-mediated mRNA decay of the ferric and cupric reductase mRNAs FRE1 and FRE2 in Saccharomyces cerevisiae. FEBS letters, 593(22), 3228-3238.

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