*2.4. Preparation of Splenocytes*

Balb/c mice (female, 8–12 weeks old, breeding facility VELAZ, Prague, Czech Republic) were used for extirpation of spleens and isolation of splenocytes. The experiments were performed according to GLP and OECD guidelines, based on the ethical guidelines of the Research Base of Slovak Medical University, Institute of Preventive and Clinical Medicine (Bratislava, Slovakia), the approval No. Ro 2939/09-221 of State veterinary and food administration of the Slovak Republic. Spleens were aseptically removed and were poured into the ice-cold saline (1 mL per spleen). Splenocytes were isolated by homogenization of splenic tissue with the plunger end of the syringe. The splenocytes suspension was filtered (50μm-mesh filter (CellTrics disposable filter; Partec, Görlitz, Germany) and centrifuged at 800× *g* for 10 min at 4 ◦C. The splenocytes were resuspended in 5 mL of ACK lysis buffer (0.15 M NH4Cl, 1 M K2CO3, and 0.01 M EDTA, pH 7.2) and the lysis of erythrocytes was completed at room temperature for 5 min. Afterwards, cells were washed twice with saline and resuspended in complete RPMI-1640 medium (Lonza, Basel, Switzerland) supplemented with 10 % of fetal bovine serum, penicillin (100 U/mL) and streptomycin sulphate (100 mg/mL) (Gibco, NY, USA). Following assessment of splenocyte viability by Trypan blue staining method, the density of cells has been adjusted to 1 × <sup>10</sup><sup>6</sup> cells/mL.
