Assessing Autophagic Flux in 2D and 3D Cell Culture Models with a Novel Plate-Based Assay
Part # PS316
Abstract
Dan F. Lazar1, Amani A. Gillette2, Braeden L. Butler1, Christopher T. Eggers1, Brock F. Binkowski1, Gediminas Vidugiris1, Michael R. Slater1, Dongping Ma1, Terry Riss1 and James J. Cali1
1Promega Corporation, 2800 Woods Hollow Rd, Madison, WI, 53711; 2University of Wisconsin, Madison WI
We have developed a homogeneous plate-based assay to measure autophagic flux that works in 2D and 3D cell culture models. The LC3 protein was tagged on its N-terminus with a spacer sequence and a small subunit of a shrimp-derived luciferase (HiBiT). When stably expressed at low to moderate levels in mammalian cell lines, this novel LC3-based reporter is processed through the autophagic pathway. The cellular level of the autophagy reporter is determined by addition of a lytic detection reagent containing a large subunit of luciferase (LgBiT) and a luminogenic substrate. LgBiT rapidly associates with HiBiT in the cell lysate producing an active NanoBiT luciferase that generates a luminescent signal proportional to the amount of autophagy reporter. Cells stably expressing the autophagy reporter and treated with stimulators of autophagy will show a decreased luminescent signal. Treatment with inhibitors of autophagy results in a buildup in the level of LC3-based reporter and thus a higher luminescent signal. The autophagic flux assay can be multiplexed (on the same sample) with a cytotoxicity assay to serve as a control to detect cytotoxic effects of test compounds. The assay has been shown to have excellent performance in an automated 384-well high-throughput screening format using U2OS and HEK293 autophagy reporter cells. The luminescent “glow” signal is stable for hours enabling batch processing of multiple 96-or 384-well plates in the same experiment. Both induction and inhibition of autophagic activity was easily observable following reference compound treatment of HEK293 cells grown as 3D spheroids.
This novel assay method enables screening for modulators of autophagic flux in 2D or 3D culture model systems using a simple homogeneous assay procedure that is recorded with a plate reading luminometer.
Printed in USA.