Interference of Microplastics with MTT and XTT Cell Viability Assays

Formazan-based assays like MTT and XTT are widely accepted and routinely employed colorimetric methods for assessing cell viability and metabolic activity in vitro. However, there is evidence that these assays are frequently subject to interference by nano- and micro-particles through various mechanisms including adsorption, abiotic reduction, and optical interference. Formazan-based assays have previously been shown to be unsuitable for applications using carbon nanotubes, metal nano- and micro-particles, and zeolite microparticles, among others. This study examines the effect of fluorescent polystyrene (PS) beads in sizes from 200 nm to 2 μm and poly-(methyl methacrylate) (PMMA) beads in sizes of 1 μm and 2 μm on the accuracy of MTT and XTT cell viability assays. Our results indicate that both PS and PMMA beads cause significant interference at sizes of 1 and 2 μm due to their attenuation spectra overlapping with the measurement wavelengths of the assays. These artifacts introduce uncertainty into viability measurements as the scattering-induced attenuation of the beads masquerades as formazan absorbance, resulting in both artificially inflated and deflated metabolic activity metrics depending on particle size and assay type. The interference of smaller 200 nm particles can be effectively corrected for by washing the plate prior to analysis. We suggest that thorough washes of the plates and additional cell-free control tests are necessary before using these assays when working with microplastics.