Flow Cytometry Analysis of Bacterial Surface Display

Bacterial cultures were grown and induced as described above. Flow cytometry analysis was performed to verify surface display of AgE6 in L. plantarum. Cells harvested from approximately 500 μl culture were washed once with PBS. The pellet was then resuspended in a 1:250 dilution of the primary antibody anti-ESAT-6 (Abcam) in PBS/2% (w/v) BSA followed by incubation for 30 min at room temperature. Subsequently, the bacteria were washed two times with 600 μl PBS/2% BSA. After the second washing step, the pellet was resuspended in a 1:170 dilution of FITC-conjugated anti-mouse IgG secondary antibody (Sigma-Aldrich) in PBS/2% BSA followed by incubation for 30 min at room temperature, protected from light. The cells were then washed three times with PBS/2% BSA before subsequent analysis using a MACSQuant analyzer (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The data were analyzed using FlowJo software. Bacterial samples used for fluorescence microscopy were prepared in the same manner and analyzed with a Zeiss LSM 700 Confocal Microscope using Zen software. Image analysis was performed using the ImageJ plugin MicrobeJ (Ducret et al., 2016 (link)). The phase contrast images were used for determination of the shape and size of the bacterial cell, and the FITC signal intensities were extracted from the corresponding fluorescent images.