2.7. Flow cytometry of mouse tissues

LNs were collected and processed for flow cytometry as described before (Commerford et al., 2018). The non‐stromal fractions obtained after pre‐digestion were pooled with one third of the stroma‐enriched fraction and used for LN macrophage and T cell analyses. Cell suspensions were resuspended in PBS and treated with anti‐CD16/CD32 (clone 93, Biolegend 101302, 1:100) for 20 min on ice before staining. EV uptake by LN stromal cells, macrophages, DCs and lymphocytes was detected using the following antibodies: rat anti‐CD31‐Fitc (BD 553372) or rat‐anti‐CD31‐APC (BD 551262) or rat anti‐CD31‐PerCp/Cy5.5 (Biolegend 102522); hamster anti‐podoplanin‐Pe (eBioscience 12‐5381‐82) or hamster anti‐podoplanin‐Pe/Cy7 (eBioscience 25‐5381‐82); rat anti‐CD45‐Pe/Cy7 (Biolegend 103113) or rat anti‐CD45‐PerCp (BD 557235) or rat anti‐CD45‐APC (eBioscience 17‐0451‐82); rat anti‐CD11b‐Fitc (Biolegend 101206) or rat anti‐CD11b‐PerCp/Cy5.5 (Biolegend 101228) or rat anti‐CD11b‐BV605 (Biolegend 101257); rat anti‐F4/80‐biotin (Biorad MCA497BB) followed by streptavidin‐Pe (Biolegend 405204) or rat anti‐F4/80‐Alexa647 (AbD Serotec MCA497A647); rat anti‐CD169‐Pe/Cy7 (Biolegend 142412); rat anti‐MHCII‐PerCp (Biolegend 107624) or rat anti‐MHCII‐Alexa700 (Biolegend 107622); hamster anti‐CD11c‐Pe/Cy7 (Biolegend 117318) or hamster anti‐CD11c‐PerCp/Cy5.5 (Biolegend 117328); rat anti‐CD8‐Fitc (Biolegend 100706) or rat anti‐CD8‐Apc/Cy7 (Biolegend 100714); rat anti‐CD4‐Pe (BD 553049); rat anti‐B220‐Pe/Cy7 (Biolegend 103222). To determine VCAM‐1 expression in LN LECs and SIINFEKL cross‐presentation, we additionally used the following antibodies: goat anti‐VCAM‐1 (R&D AF643) followed by donkey anti‐goat‐Alexa488 or donkey anti‐goat‐Alexa647 (both Thermo); rat anti‐CD45‐Apc/Cy7 (Biolegend 103116) or rat anti‐CD45‐PacificBlue (Biolegend 103126); hamster anti‐podoplanin‐Pe/Cy7, rat anti‐CD44‐BV650 (Biolegend 103049); rat anti‐Mrc1‐Pe (Biolegend 141706); and mouse anti‐H2‐K^b‐SIINFEKL‐APC (Biolegend 141606). Ki67 expression was measured using an intracellular FACS staining kit (eBioscience 00‐5523‐00) with rat anti‐Ki67‐eFluor450 (eBioscience 58‐5698‐80). To determine T cell responses in tumour‐draining LNs, we additionally used: rat anti‐CD45‐PacificBlue; hamster anti‐CD3‐Pe/Cy7 (Biolegend 100320); rat anti‐Foxp3‐Pe/eFluor610 (Thermo 61‐5773‐82), rat anti‐CD44‐BV650, rat anti‐CD62L‐Alexa700 (Biolegend 104426), rat anti‐CD25‐BV605 (Biolegend 102035), hamster anti‐CD69‐Apc/Cy7 (Biolegend 104526), AnnexinV‐Apc (Biolegend 640930); and Pe‐conjugated tetramers H‐2K^b‐SIINFEKL; H‐2K^b‐SVYDFFVWL and H‐2D^b‐EGSRNQDWL (all from NIH Tetramer Core Facility). Live / dead staining was done using Zombie‐Aqua (Biolegend 423102), Zombie‐NIR (Biolegend 423106) or 7‐AAD (Biolegend 420404). Samples were acquired using a FACS Fortessa (BD), a FACS Aria II (BD) or a CytoFLEX‐S instrument (Beckman Coulter) and the data was analysed using FlowJo v10 (BD).