Pe anti tnfα

Another male BALB/c mice were orally given 100 μL of PBS, 1.0 × 10⁹ CFU of B. longum 420, or B. longum 2012 5 times a week for 4 weeks (days 0–4, 7–11, 14–18, and 21–25) as described above. After the last oral vaccination, spleens were resected and single cell suspensions were collected. The splenocytes (2.0 × 10⁶) were cultured and re-stimulated with 2.0 × 10⁵ mitomycin-C-treated Renca cells in vitro. GolgiStop (BD Biosciences, San Jose, CA) was added to the medium after 26 h of the cell cultivation, and then the cells were cultured for additional 12 h. The cells were collected and processed using a BD Cytofix/CytopermTM Plus Fixation/Permeabilization Kit (BD Biosciences) for intracellular cytokine staining assay according to our previous studies^{4 (link),14 (link)}. As for the intracellular staining, PE-anti-IFN- γ or PE-anti-TNF- α (BD Biosciences, respectively) were used in this study. The stained cells were counted and analyzed by using Guava flow cytometer (Luminex, Austin, TX).

Freshly isolated PBMCs were stimulated with PAM and IL-2 for 12 days and the cells were cocultured with, or without, allogeneic irradiated UC-MSCs at cell ratios of 5 : 1 or 80 : 1 for 48 hours. During the last 4-hour culture, the cells were treated with Brefeldin A. Subsequently, the cells were harvested, stained with APC-cy7-anti-CD3 and APC-anti-TCRγδ, fixed, permeabilized, and intracellularly stained with FITC-anti-IFNγ, PE-anti-TNFα, PE-anti-IL-10, PE-anti-perforin, or FITC-anti-granzyme B (BD Biosciences) to examine the frequency of cytokine-expressing Vγ9Vδ2 T cells, respectively.