Cd8α fitc

The isolated PBMCs were stimulated with vaccine virus (H1N2-OH10) at 0.1 multiplicity of infection (MOI) for 48 h and immunostained for different lymphocyte subsets as described previously [14 (link)]. In brief, cells were blocked with pig serum and surface stained with pig lymphocyte-specific monoclonal antibodies. For intracellular IFNγ staining, GolgiPlug™ (BD Biosciences, San Jose, CA, USA) and Brefeldin A (Sigma-Aldrich, St. Louis, MO, USA) were added during the last 6 h of stimulation. The immunostained cells were fixed with 1% paraformaldehyde and permeabilized with cell permeabilization buffer (85.9% deionized water, 11% PBS without Ca²⁺ or Mg²⁺, 3% formaldehyde solution, and 0.1% saponin), washed and acquired 50,000 events in BD Aria II flow cytometer (BD Biosciences, San Jose, CA, USA). Using FlowJo software (Tree Star, Palo Alto, CA, USA), the data were analyzed. Fluorochrome-labeled antibodies used in flow cytometry were anti-porcine CD3 (PerCP), CD4α (APC- Cy7) and CD8α (FITC) procured from SouthernBiotech (Birmingham, AL, USA), and anti-CD8β (PE-Cy7), δ chain (APC) and IFNγ (PE) monoclonal antibodies from BD Biosciences (San Jose, CA, USA).

Cells were diluted in staining buffer (PBS 1×, 10% FBS, 0.1% Sodium Azide) and 1 × 10⁶ cells per well were transferred into 96 well-plates (V-shape), and washed twice with the same buffer. Staining was performed by resuspending the cellular pellet of each well with 100 μL of staining buffer including different combinations of antibodies, or as single-color stainings for compensation. Cells were incubated at 4 °C for 30 min and washed twice with staining buffer by centrifugation at 250 × g for 5 min.
Avian monoclonal antibodies (mAbs) (CD3-SPRD, CD4-FITC, CD8α-PE, CD8α-FITC and CD8β-PE) were purchased from Southern Biotech (Birmingham, AL, USA). Mononuclear cell suspensions from every thymus and spleen were stained with one and two mAb mix, respectively: CD3-SPRD, CD4-FITC and CD8α-PE in tube A (for thymocytes and splenocytes), and CD3-SPRD, CD8α-FITC and CD8β-PE in tube B (splenocytes). All antibodies were titrated in order to determine the optimal staining concentration of each one.
Positive cells were analyzed with a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) and CellQuest software. Analysis was done on 20 000 events and discrete viable lymphoid cell populations were gated according to the forward/side scatter characteristics. Percentages of different lymphoid cell subpopulations in the thymus and spleen were determined by multiparametric analysis.

Lymphocytes were isolated from bursal samples and were used to study T-cell infiltration by flow cytometry as previously described (Carballeda et al., 2011 (link)). Briefly, bursas were mechanically disrupted in RPMI 1640 and cellular suspensions were passed through a 40 μm mesh (Cell Strainer, BD). Mononuclear cells were isolated by centrifugation over a Histopaque density gradient. About 1 × 10⁶ cells per well were seeded on a 96-well plate and stained with different combinations of antibodies. Monoclonal antibodies (mAbs) (CD3-SPRD, CD4-PE, CD8α-FITC, Bu-PE) were purchased from SouthernBiotech (Birmingham, AL, United States). Cell suspensions were analyzed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, United States) and CellQuest software. The lymphocyte gate was defined by the forward/side scatter characteristics of the cells and 50,000 events were analyzed for each sample. Individual values of all experimental groups were normalized to the mean values of unchallenged healthy group.