Lactate dehydrogenase kit

Strains at log phase was used in the expriments. For cytotoxic assay, Caco-2 cells (1 × 10⁴)^{54 (link)} in 96 well plate was used to detect cytotoxicity of the strains (2 × 10⁵ CFU) with lactate dehydrogenase kit (Beyotime, Beijing, China). The percentage of cytotoxicity was calculated referring to the protocol of the kit: cytotoxicity (%) = (LDH release from infected cells –spontaneous release of LDH from uninfected cells) / (maximum LDH release from cell lysate–spontaneous release of LDH from uninfected cells) × 100%.
For cell adhesion and invasion, single layer Caco-2 cells in the 24 well culture plate were inoculated with 500 μL bacterial suspension (1 × 10⁶ CFU) for 3 h. After washing to remove unadhesive strain, cells were then treated by trypsin digestion for 2 h. In invasion, extracellular bacteria were treated with gentamicin (100 μg/ml) and penicillin G (5 μg/ml) before trypsin treatment. The digested cells were lysed using 1% saponin and the lysis was inoculated on THB plate. The rate of adhesion (Ra) and invasion (Ri) was expressed as (CFU _{determined from plate} / CFU _{original inoculum}) × 100%. The relative invasion rate was expressed as Ri _strain / Ri _P1/7 × 100%.

The lactate dehydrogenase kit (Beyotime, Shanghai, China) detected lactate dehydrogenase activity released by cells in different treatment groups. PC12 cells were seeded in a 96-well cell culture plate at a density of 1 × 10⁴/well, and cells were cultured according to the experimental methods in Sections “Cell culture” and “Aβ_25–35 insult, drug treatment, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay.” After the experiment, the cell culture medium of different treatment groups was collected and centrifuged (400 g, 5 min) to remove dead cells in the culture medium. Finally, the LDH in the culture medium was detected strictly according to the operation method of the kit.