Multiplexed cytometry for single cell chemical biology

Flow cytometry has great potential for screening in translational research areas due to its deep quantification of cellular features, ability to collect millions of cells in minutes, and consistently expanding suite of validated antibodies that detect cell identity and functions. However, cytometry remains under-utilized in discovery chemical biology due to the differences in expertise between chemistry groups developing chemical libraries and cell biologists developing single cell assays. This chapter is designed to bridge this gap by providing a detailed protocol aimed at both chemistry and biology audiences with the goal of helping train novice researchers. Assay users select from three elements: a small molecule input, a target cell type, and a module of cytometry readouts. For each, we explore basic and advanced examples of inputs, including screening fractionated microbial extracts and pure compounds, and target cells, including primary human blood cells, mouse cells, and cancer cell lines. One such module of cytometry readouts focuses on cell function and measures DNA damage response (γH2AX), growth (phosphorylated S6), DNA content, apoptosis (cleaved Caspase3), cell cycle M phase (phosphorylated Histone H3), and viability (membrane permeabilization). The protocol can also be adapted to measure different functional readouts, such as cell identity or differentiation and contrasting cell injury mechanisms. The protocol is designed to be used in 96-well plate format with fluorescent cell barcoding and the debarcodeR algorithm. Ultimately, the goal is to encourage the next generation of chemical biologists to use functional cell-based cytometry assays in discovery and translational research.