Black flat bottomed 96 well microtiter plates

100 µL of suspended cells was transferred to black flat-bottomed 96-well microtiter plates (Greiner Bio-One GmbH, Frickenhausen, Deutschland) and supplemented with 10 µM fluorogen of the green dye ^TFLime (ex. 480/em. 541) or the red dye ^TFCoral (ex. 516/em. 600) (The Twinkle Factory, France, Paris). Excitation and emission wavelengths both correspond to the respective maximum of the fluorogen. Fluorescence intensities of the whole population were determined using the SYNERGY H1 microplate reader (BioTek, Bad Friedrichshall, Germany) located in an anaerobic chamber and finally normalized to the OD₆₀₀ of PBS-washed cells.

Fluorescence of recombinant cells of respective strains was determined using the SYNERGY H1 microplate reader (BioTek, Bad Friedrichshall, Germany) and the Amnis® CellStream® flow cytometer (Luminex Corporation, Austin, TX, USA). Initially, cells were harvested and washed with cold PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄; 7711 × g, 10 min, 4 °C), afterwards recovered in cold PBS, and adjusted to an OD of 1. The microplate reader assay was set up by first transferring 100 µL of cell suspension of OD 1 to black flatbottomed 96-well microtiter plates (Greiner Bio-One GmbH, Frickenhausen, Germany) and supplementing with 5 μM ^TFLime (The Twinkle Factory, France, Paris). Cells were excited at 480 nm and emission determined at 541 nm. Both wavelengths correspond to the maxima of the fluorogen ^TFLime. For flow cytometry, cells adjusted to OD 1 were diluted 1:100 in pre-cooled PBS, transferred to round bottomed 96-well plates, and supplemented with 5 μM ^TFLime. 10,000 events were recorded, and fluorescence was assessed at an excitation wavelength of 488 nm using a 528/46 nm emission filter. Acquired flow cytometry data were analyzed using the CellStream™ Analysis tool version 1.2.152 (Luminex Corporation, Austin, TX, USA).