Anti cd4 v500

For flow cytometric analysis of surface markers, single-cell suspensions of lungs were incubated with a mixture containing anti-FcγRIII/II antibody (Biolegend) as well as mouse, rat, and hamster serum to block nonspecific binding. Cells were then incubated with optimal concentrations of the following specific antibodies against surface molecules: anti-CD90.2-APC-eFluor780, anti-CD127-PE-Cy7 (all from eBioscience), anti-CD44-FITC (Biolegend), anti-CD4-V500, anti-CD62L-APC, and anti-KLRG1-BV711 (all from BD Biosciences). For intracellular cytokine staining, 0.8 × 10⁶ cells were stimulated with plate-bound anti-CD3/anti-CD28 (each 5 µg/ml, BD Bioscience) or H1 (5 μg/ml) for 4.5 h in the presence of GolgiPlug™ (BD Biosciences). Cell were stained with optimal concentrations of anti-CD4-V500 (BD Biosciences), anti-CD44-FITC (Biolegend), and anti-CD90.2-APC-eFluor780 (eBioscience). Afterward cells were fixed and permeabilized with Cytofix/Cytoperm™ (BD Biosciences). Intracellularly accumulated cytokines were stained with anti-IFN-γ-PE (Biolegend) or anti-IFN-γ-V450 (BD Bioscience), respectively, anti-TNF-PE-Cy7 (Biolegend), anti-IL-17A-PerCP-Cy5.5 (eBioscience), and anti-IL-2-APC (BD Biosciences). Data were acquired on a FACSCanto™II (BD Bioscience) or on a LSRII (BD Bioscience) and analyzed with the FCS Express 5 Flow Cytometry software (DeNovo™ Software).

Single cell suspensions of spleens were prepared at the indicated time points after infection and surface markers were stained with optimal concentrations of anti-CD4-V500, anti-CD8-V450, anti-CD62L-APC, anti-CD11c-FITC, anti-CD11b-PE (all from BD Bioscience), anti-CD44-FITC, anti-Gr1-APC (both from BioLegend), anti-CD90.2-APC-eFlour780 or anti-CD90.2-PE-Cy7 and anti-MHCII(I-A/I-E)-APC-eFlour780 (all from eBioscience). T_regs were stained by using the mouse regulatory T cell staining kit (eBioscience). For intracellular cytokine staining, 1 × 10⁶ cells were stimulated with plate-bound anti-CD3/anti-CD28 (each 5 μg/ml, BD Bioscience) for 4 h or with T. cruzi antigen for 12 h in the presence of GolgiPlug™ (BD Biosciences). Cell were stained with optimal concentrations of anti-CD4-V500, anti-CD8-V450 (both from BD Biosciences) and anti-CD90.2-APC-eFlour780 (eBioscience). Afterwards cells were fixed and permeabilized with Cytofix/Cytoperm™ (BD Biosciences). Intracellular accumulated cytokines were stained with anti-IFN-γ-APC (Biolegend), anti-IL-22-PE (R&D Systems) or the isotype control rat IgG2a-PE (R&D Systems). Data were acquired on a FACSCanto^TMII (BD Bioscience) and analyzed with the FCS Express 4 Flow Cytometry software (DeNovo^TM Software). T. cruzi antigen was generated by repeated freezing and thawing of 1 × 10⁸T. cruzi parasites in 1 ml PBS.

Cell surface marker expression was analyzed by flow cytometry using a BD FACSLyric cytometer and data analysis was performed with the FlowJo software, both from Becton-Dickinson.
A surface staining protocol was applied to study the distribution of different cell subpopulations in peripheral blood. Briefly, 50 μL of peripheral whole blood were incubated with different combinations of fluorochrome conjugated monoclonal antibodies 20 min at room temperature (25°C). Red blood cells were lysed for 10 min with 2 mL of FACS Lysing solution (Becton Dickinson) and washed with phosphate-buffered saline (PBS) before flow cytometry analysis. Combinations of the following monoclonal antibodies were used: anti-CD3-PerCPCy5.5, anti-CD4-V500, anti-CD8-APCR700, anti-CD19-PECy7, anti-CD56-FITC, anti-CD45-APCH7, anti-CD45-V500, anti-HLA-DR-V450, anti-CD25-PECy7, anti-CD127-APC, anti-CD45RA-BV605, anti-CXCR5-BB515, anti-CXCR3-APC, anti-CCR6-PE, anti-CCR7-APCR700, anti-CD27-APC, anti-IgD-V450, anti-CD38-PE, anti-CD38-PerCPCy5.5, and anti-CD24-PE, all from Becton-Dickinson.