Anti cd8 v450

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.

CD8⁺ T cells were sorted and cultured as described in Sections 2.2 and 2.3. For the intracellular measurement of interferon‐γ (IFN‐γ), granzyme B (GZMB) and perforin‐1 (PRF‐1), the CD8⁺ T cell subsets were incubated with 3 μg/ml brefeldin A (Thermo Fisher Scientific, Waltham, MA) for 6 h. The harvested cells were then stained with anti‐CD8‐V450 (for details see above), anti‐IFN‐γ‐Alexa Fluor 488 (clone: B27, RRID:AB_396827), anti‐granzyme B‐Alexa Fluor 647 (clone: GB11, RRID:AB_10897997) and anti‐perforin‐1‐PE‐CF594 (clone: δG9 RRID:AB_2738410), all obtained from BD Biosciences (San Diego, CA) using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit (BD Biosciences San Diego, CA) according to the manufacturer's instructions. All antibodies were titrated to determine the optimal concentration for use. The stained samples were acquired with a FACSymphony™ A5 Cell Analyzer (BD Biosciences, Heidelberg, Germany). The FMO stainings were used for gating. The data were analysed using FlowJo software V10.7.1 (BD Life Sciences).

Peptide-MHC tetramers were generated as previously described [24 (link)]. Cryopreserved PBMC (1 million per stain or less depending on availability) from the recipient were stained with PE-conjugated or APC-conjugated peptide-MHC tetramers, anti-CD3 Pacific Orange (Invitrogen, UK), anti-CD8 V450 (BD Biosciences, UK) antibodies and near-IR Live/Dead marker (Invitrogen, UK). Samples were analysed using an LSRII flow cytometer (BD, UK) collecting a minimum of 500,000 events and gating on singlets, lymphocytes, live cells and CD3+ CD8+ cells. Data were analysed using FlowJo version 10.0.7.