Labeling, Isolation, and Analysis of Lymph Nodes

DiD-labeled LPs were injected into the foot pad of the LFM model. The ILNs and ALNs were resected at 24 h after the administration. LNs were digested in 0.1 mg/mL collagenase IV, 0.2 mg/mL collagenase D, and 0.1 mg/mL DNase I in RPMI 1640 (with 1 v/v % FBS) for 30 min. These were reliable procedures performed in the previous several reports.38 (link), 39 (link), 40 (link) The cell suspensions were washed twice with 0.5% BSA/0.1% sodium azide in PBS (fluorescence-activated cell sorting [FACS] buffer). After dispersing the cell suspensions with a 70-μm cell strainer, 2.0 × 10⁶ cells were incubated in 10 μg/μL of anti-mouse CD16/32 antibody (clone: 93; BioLegend). Cells were stained and analyzed with a Novocyte (ACEA Biosciences, San Diego, CA, USA) according to Figure S7.