An Image Cytometric MTT Assay as an Alternative Assessment Method of Nanoparticle Cytotoxicity

Abstract

Despite increasing importance of in vitro cell-based assays for the assessment of nanoparticles (NPs) cytotoxicity, their suitability for the assessment of NPs toxicity is still in doubt. Here, limitations of widely used cell viability assay protocol (i.e., MTT asssay) for the cytotoxicity assessment of P25 TiO 2 NPs were carefully examined and an alternative toxicity assessment method to overcome these limitations was proposed, where the artifacts caused by extracellularly formed formazan and light scattered by agglomerated NPs were minimized by measuring only the intracellular formazan via image cytometric methods.

The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis

“…Other than the intracellular granular form of formazan, microscopic studies have also reported formazan aggregates to appear as needle-shaped crystals on the cell surface or extracellularly. Some studies have proposed that these crystals are products of extracellular reduction of MTT by the culture media or the assessed treatment [ 42 , 43 ]; or reduction inside the plasma membrane [ 15 , 44 ]. An intracellular origin of these crystals has also been shown in many studies proposing their extrusion or extension into the extracellular space through perforating plasma membrane [ 1 ], exocytosis [ 5 , 40 , 41 ], or physicochemical processes at the molecular level [ 13 ].…”

“…As formazan absorbance is typically measured at or before 4 h of MTT incubation, the probable chemical effect of media at longer time points would not be an issue in the assay measurements. The MTT reduction by other types of culture media was also proposed including photocatalytic reduction of MTT by DMEM (Dulbecco’s Modified Eagle Medium) [ 42 ] and MTT reduction by M199 due to the presence of reducing agents such as ascorbic acid and retinol in the media [ 49 ].…”

“…There are some NPs known to catalyze redox chemistry at their solid–liquid interface and could reduce MTT [ 34 , 42 ]. To assess if our Au-PEG NPs have a similar effect, we compared the OD levels between the media solutions of the same Au-NP concentrations with and without MTT ( Figure 13 b).…”

“…Nevertheless, it has been a common practice to overlook this effect in measuring cytotoxicity or biocompatibility of Au-NPs [ 63 , 64 , 67 , 68 , 69 , 70 , 71 ], including PEG-coated or PEG-containing gold NPs/nanocomposites [ 65 , 66 , 72 ], for diagnostic or therapeutic purposes. While we have shown the confounding effect of Au-NPs on MTT assay measurements, such effects need to be considered for any other kind of treatments, as shown for TiO 2 nanoparticles [ 34 , 42 ], silicon nanowires [ 73 ], flavonoids [ 47 , 52 ], and plant extracts [ 51 ].…”

Semi‐quantitative estimation of cellular SiO2nanoparticles using flow cytometry combined with X‐ray fluorescence measurements

“…in which extraction of intracellular information from image cytometric analysis procedures has provided a unique capability to overcome one of the limitations in assessment of NPs cytotoxicity (9)(10)(11)(12)(13). Although the image cytometric approach is a novel technique that may replace current microplate reader-based toxicity assessment techniques, flow cytometry (FCM) is also one of the most powerful and widely used tools for the analysis and identification of cells in a single cell mode, which is now recognized as a simple, easy, and sensitive method, with wide applications in biological research as well as medical diagnosis.…”