2.8 | Flow cytometry
EPEC bacteria were grown overnight in LB with the appropriate antibiotics. The cultures were diluted 1:50 and grown under T3SS-inducing conditions for 3 hr. Thereafter, 1×107bacteria were plated in a 96-U shape well plate and centrifuged at 800 ×g for 5 min, and the supernatants were removed. Bacteria were incubated with primary antibody (mAb-EspB-B7, 1:100) for 1 hr at room temperature, washed with PBS, and stained using Alexa Fluor 488 goat anti-human IgG secondary antibody (Jackson ImmunoResearch) for 30 min. Samples were washed and resuspended in PBS for analysis. Flow cytometry analysis was performed on Gallios (Beckman Coulter) equipped with 488 nm, 405 nm and 638 nm lasers and a switchable 561 nm laser. Data analysis was performed with Kaluza software (Beckman Coulter).