Cd4 apc alexa fluor 780

Bone marrow‐derived dendritic cells were cultured as described¹⁹ in complete RPMI supplemented with X‐63 supernatant for 7 days. A single‐cell suspension was incubated with 10 μg/ml NP_311–325 peptide for 2 hours prior to co‐culture with lungs, spleen or lymph node cells in complete RMPI at a ratio of approximately 10 T cells to 1 DC in the presence of Golgi Plug (BD Bioscience). Co‐cultures were incubated at 37°C, 5% CO₂ for 6 hours. Cells were incubated with Fc block and surface stained with anti‐CD4 BUV805 (BD Biosciences; clone: RM4‐5) or CD4 APC‐Alexa Fluor 780 (eBioscience; RM4‐5), anti‐CD44 BUV395 (BD Biosciences; clone: IM7) and ‘dump’ antibodies: B220 (clone: RA3‐6B2), CD8 (53‐6.7) and MHC II (clone: M5114) all on eFluor‐450 (eBioscience). Cells were fixed with cytofix/cytoperm (BD Bioscience) for 20 minutes at 4°C and stained in perm/wash buffer with anti‐cytokine antibodies for 1 hour at room temperature (anti‐IFN‐γ PE (XMG1.2), anti‐TNF Alexa Fluor‐488 (MP6‐XT22) anti‐IL‐2 APC (JES6‐5H4) all from eBioscience.

A single‐cell suspension was stained with PE‐labelled IA^b/NP_311–325 (NIH tetramer core) at 37°C, 5% CO₂ for 2 hours in complete RMPI (RPMI with 10% foetal calf serum, 100 μg/ml penicillin‐streptomycin and 2 mM l‐glutamine) containing Fc block (24G2). Surface antibodies were added, and the cells incubated for a further 20 minutes at 4°C. Antibodies used were as follows: anti‐CD4 BUV805 (BD Biosciences; clone: RM4‐5) or CD4 APC‐Alexa Fluor 780 (eBioscience; RM4‐5), anti‐CD44 BUV395 (BD Biosciences; clone: IM7), anti‐CXCR5 BV785 (BioLegend; clone:L138D7), anti‐PD‐1 BV711 (BioLegend: 29F.1A12) and ‘dump’ antibodies: B220 (RA3‐6B2), anti‐CD8 (53‐6.7) and MHC II (M5114) all on eFluor‐450 (eBioscience). Cells were stained with a fixable viability dye eFluor 506 (eBioscience). In some cases, cells were then fixed with FoxP3 Transcription Factor Fixative kit (Thermo Fisher, UK) and stained with anti‐FoxP3 PeCy7 (eBioscience; FJK‐16S), anti‐Bcl2 FITC (Biolegend; Blc/10C4) and anti‐Ki67 BV605 (Biolegend; 16A8). Phosphorylated H3 was detected in cells fixed with 2%PFA/0.5% saponin using Alexa 647‐labelled anti‐Histone H3 (pS28) (HTA28, Thermo Fisher). Cells were acquired on a BD LSR or Fortessa and analysed using FlowJo (version 10 Treestar).