75 mm round bottom facs tube

Prior to FACS sorting, double emulsions were diluted 1 : 5 in FACS diluent buffer in a 12 × 75 mm round bottom FACS tube (BD Biosciences). For a typical run, 100 μL of double emulsion droplets were removed from the droplet pellet (containing high surfactant outer mix) and adding them to 500 μL of FACS diluent. Droplets were gently resuspended before analysis. See extended methods in ESI† for further discussion. Both instruments were thresholded on forward scatter, FSC, a sizing parameter, at extremely low values since DE droplets are large compared to typical cells (Table 1). Sort gates were widely permissive to show droplet purity (including sample free oil and dust, if present) compared to background. Thresholding is indicated in figure legends, if applicable. Event rates were capped below 300 events per s during sorting and 1000 events per s during analysis-only runs by modulating flow rate or flow pressure; the initial appearance of DE droplets for the Sony SH800 was typically delayed 100–200 s (see extended methods, Fig. S3†). All postprocessing analysis was completed in FlowJo v10.5.3 (FlowJo) and using custom Python scripts.