Imaging pDC-Treg Interactions in vitro

Splenic pDCs were plated at 5 × 10⁵ cells per dish in 35-mm collagen-coated glass-bottom culture dishes (MatTek) in 10% FBS complete RPMI. Cells were pulsed with PBS or low-dose or high-dose gp96 for 18 h. CD4⁺CD25⁺ Tregs or CD4⁺CD25⁻ Tconv were isolated from spleen using Treg isolation kit (Miltenyi) and labelled with CellTracker Red dye before addition to the culture and imaging. Nrp1 blocking monoclonal antibody (clone 761704; RnD Systems) or rat IgG2a isotype antibody (clone eBR2a; eBioscience) were added 2 h before imaging at a final concentration of 10 μg ml⁻¹. Antibody clones and concentrations were based on previously established protocols38 (link). Cells were imaged on a Nikon A1 inverted microscope (Nikon) using a 40X objective. Images were captured every 5 min for a total of 1 or 2 h. Videos were analysed using NIS Elements (Nikon) and ImageJ66 with Manual Tracking plugin. For interaction time analysis, total contact duration per Treg/Tconv detected from videos was calculated based on the number of frames in which a Treg/Tconv came in contact with a pDC.