Flow Cytometry of Leukocytes Subpopulations
Changes in leukocytes subpopulations were monitored by flow cytometry. One hundred microliter of EDTA treated blood was stained for 15 min in dark and room temperature by cocktail of anti-CD specific antibodies (Table antibodies, Supplemental Digital Material) at baseline, 12 and 18 h after peritonitis induction (24 h was not measured due to operational reasons). Afterward staining, all samples were lysed by BD FACS Lysing Solution (Becton Dickinson, San Jose, USA) to separate leukocytes and contaminating erythrocytes. CD14posCD16pos monocytes, T-helper (Th) and cytotoxic T (Tc) + CD8α+ γδ T lymphocytes were washed by PBS, pelleted (300 × g, 5 min) and re-suspened in 300 μl of PBS followed by measurement. T regulatory (Treg) lymphocytes were fixed and permeabilized by FoxP3 staining buffer set (Thermo Fisher Scientific), washed by PBS, pelleted (300 × g, 5 min) and re-suspened in 300 μl of PBS followed by measurement. The measurement was performed by the BD FACS Aria Fusion cell analyzer (Becton Dickinson). One million events was acquired and the post-acquisition analysis of data was performed using FlowJo software (Becton Dickinson). The gating strategy for each subpopulation is summarized in Figure 2. Absolute counts of particular subpopulations were determined from total leukocytes counts acquired within standard biochemical analysis of blood samples.
Figure 2  Particular subpopulations of leukocytes were gated from singlets populations followed by gating on FSC and SSC (A,C) and followed by gating on specific T regulatory (T reg) lymphocytes (A), on CD14+ CD16+ monocytes (B), and on T-helper (Th) lymphocytes and cytotoxic (Tc) + CD8α+ γδ T lymphocytes (C).