Rat igg2a isotype antibody clone ebr2a

On day 1, C57BL/6 mice were immunized with 10⁶ irradiated (6,000 rad) B16-OVA cells i.p. in 200 μl PBS. Seven days later, mice were given high-dose gp96 i.d. in 100 μl PBS (or PBS only as control). On the same day, mice also received 100 μg Nrp1 blocking antibody (clone 761704, RnD Systems) or 100 μg rat IgG2a isotype antibody (clone eBR2a, eBioscience) via the i.p. route. Route and dose of Nrp1 antibody is based on previous reports using this antibody38 (link). One week later, all mice were killed and draining lymph nodes were harvested for flow cytometry. The percent of Foxp3+ T cells was quantified.

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.