Flojo 7

2.5 × 10⁵ (or 1.25 × 10⁵, depending on the ALC) of viable mononuclear cells from HG or TC were stained with monoclonal Mouse anti-Chicken CD4 - FITC (clone 2–35) and CD8 - PE (clone 11–39) for T lymphocytes, or polyclonal Goat anti-Chicken IgM - FITC for B cells (AbD Serotec, UK), incubated for 30 min on ice and washed twice in PBS. The cells were analyzed with the FACSCanto II (BD, USA) flow cytometer.
Data acquisition was performed in FACSDiva Software 6.1.3. (BD, USA). Cells were analyzed and immunophenotyped in the FloJo 7.5.5 (Tree Star, USA). Relative cell counts (RCC) of T and B lymphocytes in HG and TC were established in the above environment.
Absolute cell counts (ACC) of T and B cells were calculated by dual platform analysis with the use of the following formula: ACC = (ALC * RCC)/100%. Data were expressed as the mean ACC of CD4⁺ and CD8⁺ T lymphocytes or the mean x-fold change in ACC of IgM⁺ B lymphocytes in vaccinated groups relative to control groups.

Before the experiment, the cross-reactivity of Goat anti-Chicken IgM-FITC polyclonal antibodies (AbD Serotec, UK) was checked in pigeon lymphocytes. For this purpose, mononuclear cells were isolated from the thymus, bursa of Fabricius and peripheral blood. The antibodies’ cross-reactivity was characterized by 3.99%, 44.46% and 9.5% of IgM⁺ B cells isolated from the thymus, bursa of Fabricius and peripheral blood, respectively. The quantity of the tested antibodies (3 μg antibodies per one million cells) was determined experimentally in serial dilutions (1 to 5 μg antibodies per one million cells).
Thereafter, half a million mononuclear cells isolated from spleen samples were stained with 1.5 μg of the IgM⁺ polyclonal antibody for B cells. The samples were incubated in darkness on ice for 30 min. Next, the cells were twice rinsed in PBS, centrifuged at 400 g for 10 min, and the resulting pellets were suspended in 400 μL of PBS and analyzed with the use of the FACS Canto II (BD, USA) flow cytometer. Data were acquired in FACS Diva Software 6.1.3. (BD, USA). Cells were analyzed and immunophenotyped in FloJo 7.5.5 (Tree Star, USA).