Phenotypic Analysis of Tumor-Draining Lymph Nodes

Tumor-draining LNs were pooled and digested either sequentially with 1 mg/mL and 3.5 mg/mL collagenase type IV (Thermo Fisher Scientific, Waltham, MA, USA) to enrich for LN stromal cells, as previously described [20 (link)], or directly with 3.5 mg/mL collagenase type IV in DMEM (Thermo Fisher Scientific) supplemented with 2% FBS (Thermo Fisher Scientific) and 1.2 mM CaCl₂. AccuCheck counting beads (Thermo Fisher Scientific) were added for absolute cell number determination, and anti-CD16/CD32 (101302, BioLegend, San Diego, CA, USA, 1:100) was used to block Fc receptors.
To analyze podoplanin and CD200 in tumor-draining vs. control LNs in Ackr4-GFP mice, cells were stained with CD45-PacificBlue, CD31-PerCp/Cy5.5, Podoplanin-Pe/Cy7, Mrc1-Pe, CD44-BV650, and CD200-Pe/Dazzle594. To analyze LN stromal cells in Pdpn^fl/fl x Prox1-CreER^T2 mice and control littermates, we used CD45-Apc/Cy7, CD31-PerCp/Cy5.5, Podoplanin-Pe/Cy7, CD41-Pe, Ter119-Fitc, and ESAM, followed by donkey anti-goat-Alexa647. To analyze LN macrophages, we used CD11b-BV605, CD169-Pe/Cy7, F4/80-Alexa647, MHCII-PerCp, CD80-Fitc, and CD86-Pe. Details about all antibodies are listed in Supplementary Table S1. Zombie-Aqua (BioLegend, 1:500) was used for live/dead staining. All data were recorded on a Fortessa instrument (BD Biosciences, Franklin Lakes, NJ, USA).