Phagocytosis of E. tarda by Tongue Sole PBL

Blood was collected from the caudal vein of tongue sole. PBL were isolated from the blood with 61% Percoll and collected as described previously (Li et al., 2017 (link)). The cells were cultured in L-15 medium (Thermo Scientific HyClone, Beijing, China) in 96-well culture plates (10⁵ cells/well). E. tarda TX01, TX01ΔtamA, TX01ΔtamB, TX01ΔtamA/tamA, and TX01ΔtamB/tamB were prepared as above and added to PBL (10⁵ CFU/well). The cells were incubated at 28°C for 0.5, 1, or 2 h. After incubation, the plates were washed with PBS (pH 7), and the cells were lysed with 100 μl PBS containing 1% Triton X-100. The cell lysate was diluted and plated in triplicate on LB agar plates. The plates were incubated at 28°C for 48 h, and the colonies that emerged on the plates were counted. The genetic identities of the colonies were verified by PCR with specific primers and sequence analysis of the PCR products. The experiment was performed three times.