To combat interferences seen using absorbance as endpoint readout, a cytotoxicity assay using resazurin and its fluorescent product this website was applied. AuNP-only controls suspended in EMEM medium were included and interference was detected. We observed a concentration-dependent decrease in the levels of fluorescence as a result of AuNP interference (Figure 9c). At the highest
concentration of AuNP, levels decreased by 11% to 24% depending on the AuNP in question. Au[(Gly-Tyr-TrCys)2B] exhibited the highest level of interference. The results were interpreted with care in order to avoid drawing erroneous conclusions. Cytotoxicity was assumed only when the decrease in fluorescence was lower than possible interference levels. We also examined whether the AuNPs used in this study interacted with the glutathione assay. AuNPs selleck chemicals absorbed at the wavelength used in this assay (405 nm). A dose-dependent increase appeared for some of them at concentrations of 1.56 μg/ml (data not shown) or higher. Additionally, when glutathione was incubated with a range of AuNP concentrations for 2 h the
level of free glutathione decreased as the concentration of AuNPs increased (Figure 9d). Therefore, this assay was not considered suitable for studying the oxidative stress potential of the AuNPs. However, no interference was observed with the ROS production assay (data 3Methyladenine not shown). Figure 9 PBH-capped AuNP interference with the toxicity assays. (a) MTT, (b) neutral red uptake (NRU), (c) resazurin-based cytotoxicity assay and (d) glutathione detection. Cytotoxicity Methyl thiazol tetrazolium and neutral red uptake assays The MTT and NRU assays could not be performed as there was AuNP interference at the wavelengths used in these tests (570 and 550 nm, respectively) (Figure 9a,b). Resazurin assay Cytotoxicity assays were performed Cell press with cells incubated
in EMEM/S+ and EMEM/S- after 24- and 48-h exposure periods. Only results with cells incubated in EMEM/S- are shown in Table 3, as clear evidence of cytotoxicity in cells exposed to AuNPs in EMEM/S+ could not be determined because of high interference levels in this assay under these conditions (Figure 9c). Cytotoxicity is expressed as percentage of live cells (viability) compared to the untreated control (100%). At the highest concentration (100 μg/ml), all AuNP preparations caused approximately 10% decrease in viability. This was the highest decrease in viability recorded after 24 h of incubation for the AuNP preparations tested. This decrease in viability was not higher than that recorded for the cell-free AuNP-only controls in the interference studies (11% to 24% decrease). Therefore, the reduction in viability is perceived to be a result of NP interference and cannot be reported as cytotoxicity. After 48 h of incubation, the level of cytotoxicity for Au[(Gly-Tyr-Met)2B], Au[(Met)2B] and Au[(TrCys)2B] increased significantly for the two highest doses of 50 and 100 μg/ml (p < 0.01).