Fig. 4. Droplet flow cytometry for single-cell detection. (A) Schematic diagram of rare pathogen detection by droplet-based PCR using agarose droplets and FACS. The rare pathogens (O157) and high-density normal bacteria (K12) are co-encapsulated into agarose droplets for PCR amplification. The PCR reagent mixture including two forwards primers labelled fluorescent dyes and two specific reverse primers specific for K12 and O157, respectively, were covalently conjugated to agarose. After PCR and cooling down, the agarose beads are analysed by flow cytometry for the detection of O157 cells. Adapted with permission from ref. 27. Copyright 2012 Royal Society of Chemistry. (B) Schematic workflow of the detection of cytokines secreted by single cells using agarose droplets and FC. Single Jurkat T cells were encapsulated within agarose droplets together with functionalized cytokine-capture nanobeads. After incubation, gelation, demulsification and washing, agarose beads were stained with fluorescent antibodies which can bind to the secreted cytokines, and high-throughput screening of cytokines secreted by single cells were performed by flow cytometry. Adapted with permission from ref. 25. Copyright 2013 Royal Society of Chemistry.