2.4. Phenotypic Identification of Mononuclear Cells by Flow Cytometry

Mononuclear subpopulations were monitored by flow cytometry using a panel of fluorescein (FITC) or phycoerythrin (PE)-labeled monoclonal antibodies (mAbs) for dendritic cells (DC), macrophages (Mφ), natural killer (NK) cells, B-cells, and T-cells by established methods in our laboratory [25,26] and described below.

T-Lymphocytes and Natural Killer Cells: T-lymphocytes were identified by mouse anti-pig CD3 (PE-Cy5, Clone PPT3; Southern Biotech, Birmingham, AL, USA), mouse anti-pig CD4 (FITC, Clone 74-12-4) and mouse anti-pig CD8 (PE, Clone 76-2-11) antibodies (BD Biosciences, San Jose, CA, USA) [23]. Ten microliters of each antibody were added to 1 × 10⁶ cells from each sample. Samples were incubated for 20 min and then washed twice with PBS. The immune cell phenotypes and relative abundances were assessed by BD™ LSR II flow cytometry unit (BD Biosciences). NK cells were evaluated using percentage of cells identified as CD3⁻CD4⁻CD8⁺. The relative percentage of T-lymphocyte sub-populations, single-(CD4⁺, CD8⁺) or double (CD4⁺CD8⁺) positive cells, were also evaluated using FlowJo 7.0 software (FlowJo, Ashland, OR, USA).

B-Lymphocytes: B-lymphocytes were identified using mouse anti-pig CD21 (PE, clone BB6-11C9.6, Southern Biotech, Birmingham, AL, USA) and mouse anti-pig MHCII (FITC, clone 2E9/13, ABD Serotec, Raleigh, NC, USA) antibodies [24]. Ten microliters of each antibody were added to 1 × 10⁶ cells from each sample. Samples were incubated for 20 min and then washed twice. The samples were then assessed by flow cytometry and the relative percentage of B-cells were evaluated using FlowJo 7.0 and expressed as a percentage of lymphocytes that were CD21⁺MHCII⁺. Lymphocytes, and the sub groups, were defined by FSC/SSC properties.

Dendritic Cells and Macrophages: DC were identified by mouse anti-pig CD172a (biotin, clone BL1H7, ABD Serotec; Strep-PECy5, BD Biosciences), mouse anti-pig CD16 (PE, clone G7, AbD Serotec), MHCII (FITC, clone 2E9/13, ABD Serotec) [24]. Mφ were identified by CD172a (Biotin, clone BL1H7, ABD Serotec), CD163 (RPE), CD14 (FITC, clone 74-12-4) and CD3 (PECy5) antibodies (BD Biosciences). Ten microliters of each antibody were added to 1 × 10⁶ cells from each sample. Samples were incubated for 20 min and then washed twice. The samples were then assessed by flow cytometry and the relative percentage of DC and Mφ were evaluated using FlowJo 7.0 software and expressed as a percentage of monocytes. Monocytes were defined by FSC/SSC properties, then identified as CD172a high. That population was then further separated through gating into dendritic cells (CD172a⁺, CD16⁺, MHCII⁺, CD3⁻, CD21⁻, CD163⁻) and macrophages (CD163⁺, CD172a⁺, CD14⁺, CD3⁻) and the percentage measured