Caco-2 cell was obtained from Shanghai Zhongqiaoxinzhou Biotechnology Co.,Ltd.(Shanghai, China). Cells were cultured in DMEM (Basalmedia, Shanghai, China) supplemented with 20% (v/v) fetal bovine serum (FBS, NEWZERUM, China) and 5% antibiotics (penicillin and streptomycin, Basalmedia, Shanghai, China). Caco-2 cells were seeded at a concentration of 5 × 104 cells per well in 24-well or 96-well cell culture plates to obtain a monolayer of differentiated cells.
The adhesion and invasion experiments refer to the previous studies with minor modifications (Schierack et al., 2011 (link)). After incubating A.hydrophila with EGCG for 24 hours, the mixture was centrifuged and resuspended in DMEM to adjust the bacterial concentration to 1×108 CFU/mL. The bacterial suspension was then added to a monolayer of differentiated Caco-2 cells and incubated for 1 hour. In the adhesion experiments, the culture medium was discarded, and the cells were washed three times with PBS (pH 7.4) before adding 0.25% trypsin for digestion. After gradient dilution, bacterial counting was performed. In the invasion experiments, the culture medium was discarded, and 10% antibiotics (penicillin and streptomycin) were added for 1 hour before bacterial counting was performed. LDH release was tested according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China).
The adhesion and invasion experiments refer to the previous studies with minor modifications (Schierack et al., 2011 (link)). After incubating A.hydrophila with EGCG for 24 hours, the mixture was centrifuged and resuspended in DMEM to adjust the bacterial concentration to 1×108 CFU/mL. The bacterial suspension was then added to a monolayer of differentiated Caco-2 cells and incubated for 1 hour. In the adhesion experiments, the culture medium was discarded, and the cells were washed three times with PBS (pH 7.4) before adding 0.25% trypsin for digestion. After gradient dilution, bacterial counting was performed. In the invasion experiments, the culture medium was discarded, and 10% antibiotics (penicillin and streptomycin) were added for 1 hour before bacterial counting was performed. LDH release was tested according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China).
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