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Nitroblue Tetrazolium
Published in Robert A. Greenwald, CRC Handbook of Methods for Oxygen Radical Research, 2018
Larry W. Oberley, Douglas R. Spitz
We have investigated these phenomena in great detail and have found that most of the effects are due to iron-sulfur proteins in cells.8 The bluish-purple color formation seems to be due to endogenous xanthine oxidase, while the low percentage inhibition is apparently caused by Fe-S proteins in the mitochondria. Both effects appear to be due to electron or oxygen free radical leakage from the Fe-S sites. Both effects are inhibited by thenoyltrifluoroacetone, bathophenanthrolinedisulfonic acid, disodium salt (4,7-diphenyl-1,10-phenanthrolinedisulfonic acid), and bathocuproinedisulfonic acid, disodium salt (2,9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedisulfonic acid). We have found bathocuproine to be the most effective and easiest to use inhibitor. All of these compounds will precipitate when placed in the standard assay, so bovine serum albumin (BSA) must be used to keep the compounds in solution.
The treatment of hepatocellular carcinoma with SP94 modified asymmetrical bilayer lipid-encapsulated Cu(DDC)2 nanoparticles facilitating Cu accumulation in the tumor
Published in Expert Opinion on Drug Delivery, 2023
Hao Liu, Yihan Kong, Xue Liang, Zixu Liu, Xueting Guo, Bing Yang, Tian Yin, Haibing He, Jingxin Gou, Yu Zhang, Xing Tang
Bathocuproine disulfonic acid (BCPD), a non-membrane-permeable Cu chelator, was adopted to block the cellular uptake of copper. After the cells were co-incubated with copper ions and BCPD (Cu+BCPD+DSF/DDC), the cell-killing efficiency of DSF/DDC was lower than that of copper pre-treatment groups (Pre-Cu+DSF/DDC), whereas it was still higher that of DSF or DDC monotherapy. And the IC50 of both groups were 16 μM. It was suspected that a great majority of copper ions were chelated with BCPD, whereas there was still a small part entered the cells to amplify the cytotoxicity of DSF/DDC. As revealed by the above results, copper ions play an indispensable role in DSF/DDC-based chemotherapy. When the cells take in sufficient amounts of copper ions, DSF/DDC could be more effectively complexed with copper ions to prepare the anti-cancer active substance Cu(DDC)2 in high concentration, thereby the tumor cell proliferation could be well suppressed.
Metal nanoparticles restrict the growth of protozoan parasites
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Oluyomi Stephen Adeyemi, Nthatisi Innocentia Molefe, Oluwakemi Josephine Awakan, Charles Obiora Nwonuma, Omokolade Oluwaseyi Alejolowo, Tomilola Olaolu, Rotdelmwa Filibus Maimako, Keisuke Suganuma, Yongmei Han, Kentaro Kato
Parasite culture conditions were as previously reported [8]. Briefly, Trypanosoma congolense IL3000, a savannah-type strain isolated near the Kenya/Tanzania border in 1966, Trypanosoma evansi Tansui and T. b. brucei GUTat 3.1 were maintained in the bloodstream form (BSF) in HMI-9 medium [30] and propagated at 33 °C (T. congolense) and 37 °C (T. evansi and T. b. brucei) in the air. The culture medium included Iscove’s modified Dulbecco’s medium (Sigma-Aldrich Japan, Tokyo, Japan) supplemented with 20% foetal bovine serum (FBS; Gibco, Invitrogen, Waltham, MA), 60 mM HEPES (Sigma-Aldrich, St Louis, MO), 1 mM pyruvic acid sodium salt (Sigma-Aldrich, St Louis, MO), 0.1 mM bathocuproine (Sigma-Aldrich, St Louis, MO), 1 mM hypoxanthine, 16 μM thymidine (HT supplement: Thermo Fisher Scientific K.K., Yokohama, Japan), 10 μg/L insulin, 5.5 μg/L transferrin, 6.7 ng/L sodium selenite (ITS-X: Thermo Fisher Scientific, Pittsburgh, PA), 0.0001% 2-β-mercaptoethanol (Sigma-Aldrich) and 2 mM L-cysteine (Sigma-Aldrich, St Louis, MO). The cultures were maintained by replacing the entire supernatant with fresh medium every other day.
Impediment to growth and yeast-to-hyphae transition in Candida albicans by copper oxide nanoparticles
Published in Biofouling, 2020
Alwar Ramanujam Padmavathi, Sriyutha Murthy P., Arindam Das, Arumugam Priya, T. J. Sushmitha, Shunmugiah Karutha Pandian, Subba Rao Toleti
Copper ions internalized by C. albicans cells were estimated by the Bathocuproine assay with a minor modification (Koga et al. 2019). Bathocuproinedisulfonic acid disodium salt (BCS) is a Cu+ chelating agent that forms a strong complex with monovalent copper ions (Supplementary Figure S1). Cu(I)-BCS complex has a typical orange colour that has maximum absorption at 485 nm. Cu++ can also be measured by reducing into Cu+ using a strong reducing agent. BCS solution was prepared in an amber coloured bottle by dissolving 0.36 g of BCS in 100 ml of 10 mM phosphate buffer (pH 7.4). Neutralization buffer was prepared by mixing 60 ml of 1 M acetic acid and 25.2 ml of 1 M NaOH. Ascorbic acid (AA) (0.5 mM) was used as a reducing agent. C. albicans was grown in the absence (control) and presence (treated) of 50 µg ml−1 CuO-NP and Cu2O-NP separately at 37 °C for 24 h. After incubation, cells were pelleted down and washed thrice with PBS. The cells were centrifuged at 5,000 rpm for 5 min and the cell pellet was suspended (OD 1.0) in PBS. A 3 ml cell suspension was subjected to probe sonication to release the internalized copper ions. Cell debris was removed by centrifugation (6,000 rpm, 5 min) and 22 µl of supernatant were mixed with 2.5 ml of neutralization buffer and 219 µl of BCS solution and stirred continuously under dark for 20 min. For measuring internalized divalent copper ions (Cu++) arising from the CuO-NP treatment, an equal volume of AA was added to the supernatant and incubated at room temperature prior to the addition of BCS solution. The absorption of the Cu(I)-BCS complex was measured at 485 nm and the Cu(I) concentration was calculated using the following formula: 4 at 485 nm), 125 is the dilution factor.