Flow Cytometry Analysis of Double Emulsions

Double emulsions were resuspended with a 200 μL
pipet prior
to measuring. Flow cytometric analysis was carried out on a CytoFLEX
S machine for double emulsions stored in TAB (1.5% tween). Aptamer-Cy5
fluorescence was quantified using 640 nm excitation, with a 660/10
nm bandpass filter. Flow cytometric sorting of double emulsions was
performed on FACSAria III or FACSAria II instruments (BD) , with sorting
into different low-binding tubes (Eppendorf) containing 100 μL
of nuclease-free water according to aptamer fluorescent intensity.
Prior to sorting, the double emulsions were often diluted ∼5
times in TAB (1.5% tween). Cy5 fluorescence was quantified using 633
nm excitation, with a 660/20 nm bandpass filter. Importantly, the
nozzle size to smoothly accommodate the 22.5 μm double emulsion
droplets was 130 μM. The selection threshold was deliberately
set close to the level of background noise, to create relatively permissive
screening conditions and avoid missing moderately improved catalysts
in library screens.^{49 (link),53 (link)}