High-throughput cell-based fluorescent imaging assays often require removal of background fluorescent signal to obtain robust measurements. Processing high-density microplates to remove background signal is challenging because of equipment requirements and increasing variation after multiple plate wash steps. Here, we present the development of a wash-free cell-based fluorescence assay method for high-throughput screening using a laser scanning fluorescence plate cytometer. The cytometry data consisted of cell count and fluorescent intensity measurements for phenotypic screening. We obtained robust screening results by applying this assay methodology to the lysosomal storage disease Niemann-Pick disease type A. We further demonstrated that this cytometry method can be applied to the detection of cholesterol in Niemann-Pick disease type C. Lastly, we used the Mirrorball method to obtain preliminary results for the detection of Zika and Dengue viral envelope protein. The advantages of this assay format include 1) no plate washing, 2) 4-fold faster plate scan and analysis time, 3) high throughput, and 4) >10-fold smaller direct data files. In contrast, traditional imaging assays require multiple plate washes to remove the background signal, long plate scan and data analysis times, and large data files. Therefore, this versatile and broadly applicable Mirrorball-based method greatly improves the throughput and data quality of image-based screening by increasing sensitivity and efficiency while reducing assay artifacts. SIGNIFICANCE STATEMENT: This work has resulted in the development of broadly applicable cell-based fluorescence imaging assays without the requirement of washing out reagents to reduce background signal, which effectively decreases the need for extensive plate processing by the researcher. We demonstrate this high-throughput method for drug screening against lysosomal storage diseases and a commonly used viral titer assay.
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