Nb100 725b

Anti-β-actin (1:1,000, A5441, Sigma-aldrich), anti-p-Tyr antibody (1:500, sc-7020, Santa Cruz), anti-VEGF-C antibody (1:500, sc-374628, Santa Cruz), anti-VEGFR3 antibody (1:500, sc-365748, Santa Cruz), anti-iNOS antibody (1:500, ab3523, Abcam), anti-IL-1β antibody (1:500, ab9722, Abcam), anti-TNF-α antibody (1:200, GTX110520, GeneTex), anti-TGF-β antibody (1:200, ab64715, Abcam), anti-CCL28 antibody (1:500, MAB717, R&D systems), anti-Podoplanin antibody (1:200, sc-166906, Santa Cruz), anti-CD31 antibody (1:200, 550274, BD biosciences), anti-LYVE-1 antibody (1:500, NB100–725B, NOVUS biologicals), anti-vWF antibody (1:100, A0082, Agilent), FITC anti-CD4 antibody (1:100, 561828, BD Biosciences), FITC Neutrophil antibody (1:100, ab53453, Abcam), PE anti-CD34 antibody (1:100, 551387, BD Biosciences), APC anti-F4/80 antibody (1:200, 123116, BioLegend), FITC anti-CD16/32 antibody (1:200, 101306, BioLegend), PE anti-CD11b antibody (1:200, 557397, BD biosciences), anti-CD45 antibody (1:400, 20103–1-AP, Proteintech).

Primary CLN lymphatic endothelial cultures were prepared from CLNs of C57Bl/6J mice (12–14 weeks). Briefly, CLNs were dissected, then cell suspensions were prepared. Biotin-conjugated LYVE-1 antibody (10 μg, NB100-725B, NOVUS biologicals) was mixed with CELLection Dynabeads (11533D, Thermo Fisher Scientific) coated with recombinant streptavidin (100 µl, 4 × 10⁷ beads) for 30 min. After removing free floating antibody, beads were mixed with cell suspensions for 20 min at 4 °C. After the process of collecting the beads-bound cells and unbinding the beads, cells were plated and cultured with EBM2 plus supplement growth factors for 5–7 days.