2.1. PBMC Culture and Stimulation

PBMC were isolated from the whole blood drawn from seven adult roosters of Green-legged Partridgelike (GP). Whole blood was collected from the wing vein to a tube containing heparin as an anticoagulant. The protocols were approved by the Local Ethics Committee for Animal Experiments (Bydgoszcz, Poland) (study approval reference number 16/2014). Heparinized blood was diluted with HBSS (without Ca²⁺ and Mg²⁺) 1:1 ratio. A volume of 20 mL of diluted blood was transferred to the falcon tube containing 20 mL of 1.077 Histopaque. The falcon tube was centrifuged (centrifuge 5810R, Eppendorf, Hamburg, Germany) at 260× g for 30 min at room temperature (24 °C). The PBMC layer was drawn and transferred to a new Falcon tube. Fresh HBSS (without Ca²⁺ and Mg²⁺) was added up to the volume of 30 mL. The falcon tube was again centrifuged at 425× g for 20 min. The supernatant was removed, and the cells were rinsed with 20 mL of the fresh HBSS (without Ca²⁺ and Mg²⁺). The suspended PBMC were centrifuged again at 425× g for 5 min. The supernatant was removed and the cells were resuspended in 30 mL of RPMI 1640 medium (Biological Industries, Beit Haemek, Israel), supplemented with 10% FBS (Biological Industries, Beit Haemek, Israel), 1% GlutaMAX (Gibco, Dublin, Ireland), and 1% Antibiotic-Antimycotic solution (Gibco, Dublin, Ireland). PBMC were pre-incubated in a culture flask on a shaker for 3 h in a humidified incubator at the temperature of 41.5 °C and atmosphere enriched in 5% CO₂. After pre-incubation, PBMC were scraped from the flask and transferred to the falcon tube. Cells were centrifuged at 425× g for 5 min and resuspended in fresh RPMI 1640 media with supplements as described above (but without 1% Antibiotic-Antimicotic solution). Countess Automated Cell Counter (Invitrogen, Carlsbad, CA, USA) was used to count cells. Viability was estimated using differential cell staining with 0.4% trypan blue (Invitrogen). PBMC were diluted to the working concentration of 1.25 × 10⁷/mL and distributed on 6-well plates (2 mL/well). Cells were incubated overnight prior to stimulation at 42.5 °C in 5% CO₂. On the day of stimulation, a volume of 100 µL of the respective stimulus was added to the well and mixed by gentle pipetting. The control wells were mock-stimulated with the media. The list of the stimuli is shown in Table 1. PBMC stimulation was conducted in a time-course manner; the cells were harvested at 3 h, 6 h, and 9 h post-stimulation. The experiment was repeated three times (biological replicates) at one-week intervals.

Toll-like receptor (TLR) ligands and live probiotics are used to stimulate chicken peripheral blood mononuclear cells (PBMC) in vitro.