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A Novel, Bioluminescent Assay for the Selective Detection of Target Cell Killing in Mixed Cultures

Part # PS358

Abstract

Brock Binkowski, Aileen Paguio, Peter Stecha, Christopher Eggers, Braeden Butler, Michael Beck, Julia Gilden, Zhi-Jie Jey Cheng, Mei Cong and Frank Fan Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711

Efforts to develop cellular immunotherapies would benefit from assays that selectively monitor target cell death that are sensitive and easy-to-use. To address this, we have developed an approach to selectively quantify target cell death using a gain-of-signal assay format and bioluminescence read-out. The method relies on the release of a HiBiT-tagged protein from target cells following cell killing. HiBiT, an 11 a.a. peptide tag, binds to cellimpermeable Large BiT (LgBiT), a 17.6 kDa protein, to reconstitute NanoBiT Luciferase. Target cells are engineered to express a HiBiT-tagged protein using either ectopic expression or CRISPR/Cas9 to tag endogenous LDH, and cell lysis is quantified by adding a detection reagent containing LgBiT and furimazine substrate (no medium removal). The signal is proportional to the amount of target cell death, and measurements can be made using endpoint or kinetic formats.

Printed in USA.