Reactive oxygen species assay

Selected entries from Methods in Enzymology [vol, page(s)] Assay with 2,2-dithiobisnitrobenzoic acid method, 233, 381-382 pH effects, 233, 384-385 formed by incubation with dithiothrei-tol, labeling, 233, 409-410 protein thiol assay, 234, 273-274 labeling, 233, 414 reactivity with free radicals and reactive oxygen species, 233, 405 reaction with ferrylmyoglobin, 233, 196-197. 

AP-induced toxicity and measurement of cell survival/injury were performed as described in detail elsewhere.1011 Briefly, 6-day-old cells were exposed to fresh solutions of either Ap25 35 or Ap, 42 for 24 h, in the presence or absence of different drugs. Cell survival and extent of cell death were determined using MTT and Sytox green assays, respectively. Measurement of intracellular reactive oxygen species was determined by dichlorofluorescein (DCF) fluorescence assay, as described previously.23... 

Since free radical accumulation was proposed to mediate Ap toxicity,27 we studied the effects of EGCG on intracellular reactive oxygen species (ROS) using DCF assay. As expected, a 24-hour exposure to Ap, 42 resulted in a small but significant increase in DCF fluorescence (+18% relative to control), which was reduced by EGCG (10 xM) and by EC (101xM), a tea catechin that failed to protect cells. 

G10. Glazer, A. N., Phycoerythrin fluorescence-based assay for reactive oxygen species. Meth. Enzymol. 186, 161-168 (1990). 

In 2005, Lopez de Cerain et al. found that by using comet assays and flow cytometry analysis, compound 43 (Table 6) damages DNA under hypoxia and normoxia conditions [150]. In another study, it has been demonstrated that compound 43 produces reactive oxygen species (ROS) and oxidative DNA damage both in normoxic and hypoxic conditions into Caco-2 cells [151]. In normoxia, a significant increase of ROS was evidenced in a manner dose-dependent, while in hypoxia high ROS levels were observed at all the studied concentrations. Furthermore, modified comet assay demonstrated that oxidative DNA damage in normoxia and hypoxia was promoted by compound 43. 

In addition to measuring the antioxidant effects of prenylated flavonoids from Sophora flavescens by using in vitro l,l-diphenyl-2-picrylhydrazyl (DPPH), 2,2/-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), per-oxynitrite (ONOCT), and total reactive oxygen species (ROS) assays, the examination of the inhibition of tert-butylhydroperoxide (f-BIIP)-induced intracellular ROS generation and f-BHP-induced activation of nuclear factor-kB (NF-/cB) was also added as a further examination of kuraridinol (9),... 

The development of serum-free medium for fish cell lines has had some success3 20 and could be used to overcome the difficulty of toxicant availability3. In addition, FBS contains protective molecules, such as antioxidants, which could inhibit death elicited by reactive oxygen species (ROS). This means that basal medium can be more appropriate for acute assays and even simpler exposure solutions might be best. Schirmer et al.115 used an extremely simple one, termed L-15 exposure or L-15/ex. L- 15/ex contains only salts, galactose and pyruvate at their concentrations in the basal medium, Leibovitz s L-15. The expression of cytotoxicity appeared to be aided by the absence in L-15/ex of most antioxidants. Also, for photocytotoxicity studies, the lack of vitamins and aromatic amino acids prevented the inadvertent generation of toxicants from some of these compounds during the UV treatment. 

The reactive oxygen species production, stimulated by PMA, essentially due to the activity of NADPH oxidase system, was adopted as cellular functional character in human HL-60 cell line. R.O.S. metabolism was studied by CL assay as described. Assays were performed in triplicates at 25 °C. CL system contained in 1.00 mL final volume with modified KRP solution 100 nmoles luminol, 1 x 10 cells without treatment (control) or treated with ATRA or monomers, in presence or absence of... 

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