Fix perm cell fixation and cell permeabilization kit

MDCK, MDCK-FcγRI and MDCK-CTLA4 cells were trypsinized and washed with FACS buffer (1× PBS containing 1% bovine serum albumin (BSA), 2 mM EDTA, 0.02% sodium azide, and HEPES). Cells were incubated in blocking buffer (PBS containing 4% goat or rabbit serum, 1% BSA, 0.02% sodium azide) and stained with anti-CTLA4 Nbs and cHcAbs. Bound Nbs or cHcAbs were detected by using anti-His 647 or anti-canine IgG Fc 647 or 750 secondary antibodies and analyzed by flow cytometry. Canine PBMC were cultured overnight in RPMI media. PBMCs were stimulated with various concentrations (50, 100 and 150 ng/mL) of PMA and Ionomycin (1 µM/mL final concentration) for 6 h. Stimulated PBMCs were blocked and stained with anti-CTLA4 Nbs and cHcAbs. Cells were also stained with anti-CD3FITC (clone CA17.2A12), anti-CD4RPE (clone YKIX 302.9), and anti-CD8 647 (clone YCATE55.9) antibodies to characterize subpopulations expressing canine CTLA4. For intracellular FOXP3 staining, cells were fixed and permeabilized using the fix & perm cell fixation and cell permeabilization kit from Life Technologies and stained with FOXP3 monoclonal antibody (FJK-16s [ThermoFisher]).

Tonsillar tissues were colonized with 15 vaginal Lactobacillus strains (27 blocks per condition) at a starting inoculum of 10⁴ CFU/mL. At day 3 after inoculation with bacteria, all tissue blocks were collected and digested with collagenase IV (5 mg/mL; Gibco BRL) for 30 min with agitation in a Thermomixer at 900 rpm at 37°C. Following digestion, tissue cells were filtered with 100 μm cell strainers (Corning) and washed with 50 mL of PBS. Cells were then suspended in 1 mL of PBS and stained with 1 μl of live/dead Fixable Viability Dye eFluor 450 (EF 450, Invitrogen) for 15 min. After incubation, cells were washed and diluted in staining buffer [PBS, 1% normal mouse serum, 1% normal goat serum, 1 mM ethylenediaminetetraacetic acid (EDTA)] and stained with anti-CD3-allophycocyanin (CD3-APC) for 20 min. After surface staining, cells were permeabilized with the Fix&Perm Cell Fixation and Cell Permeabilization Kit (Invitrogen) then stained for 20 min with anti-Bcl2-PE, a mitochondrial anti-apoptotic antigen. Data were acquired with a Novocyte flow cytometer (ACEA Biosciences, CA, United States) equipped with 405, 488, and 640 nm laser lines using NovoExpress version 1.2.4 software (ACEA Biosciences) and analyzed using the same software.