ssary to reliably define hit compounds in the context of a highthroughput screen using the assay conditions reported in this study. Notably, TFEB overexpression resulted in a 3-fold increase in NAG activitythus much above the threshold required to define a reliable hit, which would make TFEB a strong candidate in a genetic screen. Based on the results obtained, we can conclude that the NAG 96-well plate assay has desirable characteristics of sensitivity and reproducibility that make it suitable for high-throughput screening applications. Discussion In this study, we present a rapid, reliable and robust assay to measure NAG activity in a 96-well plate format that complements existing methods and presents desirable characteristics that make it particularly attractive for primary screening in high-throughput applications. The NAG assay herein described, in fact: involves a reduced number of steps, resulting in a shorter protocol; requires a reduced number of cells, enabling the use of the 96-well plate format, which, in turn, allows testing multiple mutations, culturing and treatment conditions simultaneously and with a NAG One-Step Cell Assay higher number of replicates; is performed in a single plate from start to finish, with no requirement for transfer of samples or material across plates; benefits from reduced protein inactivation due to denaturation or degradation, thus resulting in more reproducible results. On the other hand, existing methods to measure 15276073 NAG activity require several steps of sample preparation that include cell disruption by sonication or repeated cycles of freeze-thawingsteps that are time consuming and difficult to standardize because they may result in protein denaturation or incomplete cell lysis, thus impacting the measured enzyme activity. As a result, the amount of cells and time typically needed for each sampleincluding normalization of the readout signal by DNA or protein contentare hardly compatible with a systematic testing of multiple cell lines or conditions. In the NAG 96-well plate assay, the same number of cells is plated in each well and the number of steps involved in the experimental procedure is kept at a minimum to minimize differences in readout values due to differential cell number or growth rate. Assays conducted using 13 fibroblast lines derived from MPS IIIB patients showed that the readouts were uniformly,20-fold lower than control fibroblasts from healthy donors in all cases, which supports the reliability of the assay to assess deficiencies in NAG activity. In addition, upregulating NAG synthesis in 19497313 HeLa cells by inducing the activation of the master lysosomal regulator, TFEB, via sucrose treatment or by direct TFEB transfection allowed evaluating the assay sensitivity to increases in NAG expression and activity. The readouts of the assay showed that changes in NAG activities paralleled changes in NAGLU mRNA expression as detected by 2883-98-9 web real-time qPCR. Together, these data support the notion that the assay is run in conditions that are well above the background noise of the analysis and in a range of values that is far from the saturation of the signal. An assessment of reproducibility showed that the Z9 score associated with the NAG 96-well plate assay was higher than 0.6 in experiments where sucrose-mediated increase in NAG activity averaged 1.7-fold. Subsequent calculations that took into account our observed standard deviation of,5% showed that an increase in NAG activity $1.6-fold wou