Analytical Chemistry Live/Dead (calcein/EthD-1) Assay. The Live/Dead Viability/ cytotoxicity Kit was used with final concentrations of 10 μM Calcein or 20uM EthD-1 added to the appropriate volumes of dUSPION in a 96-well plate. The fluorescence was read after 1 h. For calcein fluorescence, the excitation and emission wave- lengths utilized were 485 and 530 nm respectively, while for EthD-1, the wavelengths were 530 and 645 nm. The principle of using calcein is that the cell’s ubiquitous esterase activity converts the virtually nonfluorescent, cell-permeant calcein AM, to the highly fluorescent calcein, which is retained within the cell. On the other hand, EthD-1 is used to detect dead cells as it cannot enter through the intact plasma membrane of live cells; it can, however, easily enter damaged cells. Upon binding to cellular nucleic acids, EthD-1 increases in fluorescence intensity 40-fold producing a bright red fluorescence detected at 635 nm. DCFH-DA Assay. DCFH-DA assay is based on the principle that upon internalization, the diacetate (DA) portion of the hydrophobic dye is cleaved by intracellular esterases. The result- ing DCFH is nonfluorescent until it is oxidized by ROS to its highly fluorescent product DCF. Initiation of the DCFH-DA assay requires this DA portion of the molecule to be cleaved, and in acellular systems, this cleavage can be achieved via chemical means using sodium hydroxide (as in the present study) or using media. The excitation of the DCF molecule at 485 nm emits green fluorescence at levels proportional to the amount of ROS present, which can be detected at 520 nm. DCFH-DA was dissolved in DMSO to a concentration of 1 M and was further diluted to the appropriate concentration with Hepes-buﬀered (20 mM) Hanks balanced salt solution with glucose (5 mM) (pH 7.4). Before using DCFH-DA in a cell free system, chemical cleavage of the diacetate (DA) portion was necessary by incubation with 10 mM NaOH in the dark, at room temperature, for 30 min. The resulting DCFH was neutralized with 25 mM phosphate buﬀer (1:1 sodium phosphate monobasic: sodium phosphate dibasic) (pH 7.4) and the solution was kept in the dark, on ice until use. A 2 μM portion of DCFH was then added to the wells of a 96-well plate previously loaded with dUSPION, and ﬂuorescence was measured over 1 h with ﬂuorescence excita- tion and emission at 480 and 520 nm, respectively. To determine if the increases in ﬂuorescence signal observed with maghemite was indeed due to the generation of ROS induced by dUSPION (as opposed to dUSPION interaction with assay components exclusively), further experiments were performed in the presence of 2 mM NAC, which was applied to the plates with dUSPION, prior to DCFH. The concentration of NAC used (2 mM) was chosen, as preliminary studies showed this concentration to be suﬃciently potent to reduce dUSPION induced increases in DCFH signal. Also, to determine whether the decrease in ﬂuorescence observed with magnetite was due to interactions with DCFH or whether magnetite actually has antioxidant properties, experiments were done to see whether the presence of magnetite can prevent or reverse TBHP-induced oxidative stress. For this, TBHP (25 mM) was applied to the plates with dUSPION and compared to wells loaded with TBHP alone, to look for a reduction in ﬂuorescence. For all DCFH experiments, because of the dynamics of the DCFH ﬂuorescence over time, time zero readings were sub- tracted from time 60 min readings. APF Assay. APF was added to a dUSPION preloaded 96-well plate giving a final concentration of 8 μM, and fluorescence was measured over 1 h (fluorescence excitation and emission at 480 and 520 nm, respectively). ARTICLE Table 1. Characterization of dUSPION: Summary of the Physicochemical Features of the dUSPION Assessed under Experimental Conditions maghemite magnetite diameter (DLS; nm) In RPMI-1640 with 1% serum In hepes-buﬀered HBSS with glucose zeta-potential (mV) XPS ratio (Fe2þ/Fe3þ) 80.3 ( 6.0 91.0 ( 31.9 11.4 ( 2.5 0.118 143.2 ( 11.5 128.3 ( 2.0 12.0 ( 1.6 0.435 Statistical Analysis. A one-way ANOVA with a two-sided Dunnett’s post hoc test was performed for each data point (n = 3) comparing each one to its relevant untreated control. Fisher’s exact test was used to compare each dose of dUSPION treatment with NAC to its relevant zero NAC control. For all graphs, data is presented as percentage of control without dUSPION inclusion. ’ RESULTS AND DISCUSSION Several recent studies have investigated the potential of engi- neered NM to induce cytotoxicity, oxidative stress, and genotoxi- city. However, results from these studies are not always consistent, and consequently, a great degree of uncertainty regarding the true toxicity of NM still exists.1,2,4,23 28 One potential explanation for this uncertainty is the lack of standardized protocols and a deﬁciency in appropriate controls when using certain assays for studying the toxic eﬀects of NM.23,28 30 We have previously shown that standard DNA damage assays also need modiﬁcation when dealing with NM as opposed to chemicals for which they were originally optimized.28 When considering the best approach for characterization of NM, it must be recognized that due to their unique physicochemical properties it cannot be assumed that NM can be tested in the same way as chemicals and that there may be some confounding factors skewing the results, which may result in misinterpretation of data sets. In fact, previous studies have shown that carbaceous NM and nanotubes interact with a range of colorimetric and ﬂuorometric probes used for testing cytotoxicity and oxidative stress, including MTT, neutral red, IL-8 cytoset ELISA, almar blue, WST-1, and Coomasie blue assays, and is thought to be due to the adsorbing properties of NM resulting in false readings.11 13,31 34 Interactions between NM and assay components are particu- larly problematic when these test systems are central to assessing NM safety. Thus, where colorimetric and ﬂuorometric dyes are to be relied on for experimental test systems, potential alteration of background signal due to interference imparted by the NM must be considered, the importance of which is demonstrated in the present study using dUSPION. The physicochemical features of dUSPION were assessed under experimental conditions (Table 1), and as shown in Figure 1, both maghemite and magnetite dUSPION were spherical with a core diameter of ∼10 nm. However, the latter dUSPION exhibited a slightly more pronounced degree of agglomeration. When using the MTS assay in a cell-free system, both dextran- coated maghemite and magnetite showed no signiﬁcant change in absorbance levels, between concentrations of 1 10 3 and 10 μg/mL as illustrated in Figure 2a. However, 100 μg/mL of both maghemite and magnetite dUSPION samples caused a signiﬁcantly dramatic (9.5- and 6.5-fold, respectively) increase 3780 dx.doi.org/10.1021/ac200103x |Anal. Chem. 2011, 83, 3778–3785