Anti-TGEV Miller Strain Infection Effect of Lactobacillus plantarum Supernatant Based on the JAK-STAT1 Signaling Pathway

Wang, Kai; Ran, Ling; Yan, Tao; Niu, Zheng; Kan, Zifei; Zhang, Yiling; Yang, Yang; Xie, Luyi; Huang, Shilei; Yu, Qiuhan; Wu, Di; Song, Zhenhui

After centrifugation of Lp-1 with an OD 600 of 1.8 at 4000 rpm/min for 10 min, the obtained supernatant was filtered through a 0.22-µm filter, and then diluted with RPMI 1640 high sugar medium to six gradients at two times ratio, i.e., Lp-1s was diluted to obtain OD 600 values of 0.9, 0.45, 0.225, 0.113, 0.056, and 0.028, respectively. IPEC-J2 cells were seeded at 1 × 10 5 /mL in 96-well plates and incubated overnight in 5% CO 2 at 37 • C. The medium was discarded when the cells reached 90% confluence in the 96-well plate, and then 100 µL of each gradient dilution of Lp-1s was added to each well. The medium in the control group was replaced with RPMI 1640. After incubating for 90 min, the supernatant was discarded, and the cells were washed twice with phosphate-buffered saline (PBS). Culture was continued and the cytopathic effect (CPE) was observed daily. The maximum non-toxic dose of Lp-1s to the cells was detected using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (BBI, Shanghai, China), when 80% of the cells in the negative control group were damaged. MTT assays for each dilution were repeated three times independently.