Ldh release

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 viability of HaCaT cells was detected by MTT method, consistent with our previous research [23 (link)]. In addition, lactate dehydrogenase (LDH) release was also used to analyze the cytotoxicity, according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The LDH release was detected at 490 nm wavelength by a microplate reader.

Cytotoxicity assessed by the 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay and LDH release (all from Nanjing jiancheng, Nanjing, China). The MTT Assay: briefly, the aliquots containing 5 × 10⁵ cells/mL were plated in a 96-well plate. The cells were incubated at 37 °C for 72 h. MTT (0.5 mg/mL) was added to each well at 4 h before the end of the time point, then the MTT reaction was inhibited by addition of 10% SDS and 0.1 M HCl. The formazan crystals formed were dissolved in DMSO, and the absorbance was measured at 490 nm in a microplate reader (Bio-Tek ELX800, Bio-Tek Instrument Inc., Winooski, VT, USA). The LDH assay was performed according to the manufacturer’s guidelines. The percentage of LDH release was calculated by the equation. LDH release (%) = (Experimental LDH release − spontaneous LDH release)/maximum LDH release.