Quantification of Stx1B and CNL in L. lactis

L. lactis cultures (20 μL) in the stationary growth phase were added to 500 μL of TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) and centrifuged at 5000× g for 5 min at 4 °C. The pellets were resuspended in 500 μL of TBS containing rabbit polyclonal anti-FLAG-tag (1:500; Proteintech) or anti-CNL (1:1000) for the detection of Stx1B and CNL, respectively [43 (link)]. After 2 h of incubation at room temperature (RT) with constant shaking at 100 rpm, the cells were washed three times with 200 μL of TBS with 0.1% Tween-20 (0.1% TBST) and resuspended in 500 μL TBS containing an Alexa Fluor 488-labelled anti-rabbit antibody (1:2000; Cell Signaling Technology). After 2 h of incubation at RT with constant shaking at 100 rpm, the cells were washed three times with 200 μL of 0.1% TBST and finally resuspended in 500 μL TBS. The samples were analyzed using a flow cytometer (FACS Calibur; Becton Dickinson, Franklin Lakes, NJ, USA) with excitation/emission at 488/530 nm in the FL1 channel. The geometric mean fluorescence intensity of at least 20,000 L. lactis cells in the appropriate gate was measured.