Cytofix cytopermtm plus fixation permeabilization kit

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).

Activated PBMCs were stained with anti-BDCA4-APC (CD304, Neuropilin-1, BioLegend, Cat. No. 354506) for 30 min at 4 °C then were fixed and permeabilized with BD Cytofix/CytopermTM Plus Fixation/Permeabilization Kit (BD Biosciences) according to the manufacturer’s instructions. After washing cells were incubated with anti-cleaved IL-1β (Asp116; clone D3A3Z; Cat. No. 83186S) or anti-cleaved caspase-1 (Asp297; clone D57A2; Cat. No. 4199S) antibodies (all from Cell Signaling) for 30 min at 4 °C. After washing cells were incubated with PE-conjugated IgG1 secondary antibody (Cat. No. 406421; BioLegend) for 30 min at 4 °C. As an isotype control rabbit IgG (Cell Signaling, clone DA1E, Cat. No. 3900S) was used. Stained cells were dissolved in Mowiol^® 4–88 mounting media (Sigma-Aldrich, Cat. No. 81381).
For confocal microscopy 100,000 cells were pipetted on 8-well μ-Slides (chambered coverslip, ibidi GmbH). Cells were visualized immediately by a Zeiss LSM880 confocal microscope. BDCA4-APC was excited at 633 nm and IgG1-PE was excited at 543 nm. Fluorescence emission was detected through 650 to 670 nm and 560 to 615 nm band-pass filters. Images were taken in multi-track mode to prevent cross-talk. Image stacks of 1024 × 1024 pixel, 1.5 μm thick optical sections were obtained with a 40× C-Apochromat water immersion objective (NA = 1.2).