Fig. 4. Tracer distribution to LN-localized cell subsets varies between intravenous versus intradermal routes of administration. (A) Experimental workflow flow cytometrically quantifying tracer association with leukocyte subsets within LNs. (B) Representative flow cytometry plots of fluorescent tracer associated with leukocytes in axillary (AX) and brachial (BR) LNs collected from mice either i.d. or i.v. administered 30 nm PPS NP. LNs collected from mice not administered tracer were used to define tracer positive signal above background in experimental samples. (C) Number of cells within the LN associated with tracer from i.v. and i.d. administration. *, ** indicate significant difference (p < 0.05, p < 0.01, respectively) between i.v. and i.d. administration for each tracer size as determined by Welch ANOVA. Data are presented as mean ± SD. (D) Number of SSM (CD3⁻B220⁻CD11b⁺CD169⁺F4/80⁻CD11c^LO) within the LN associated with tracer from i.v. and i.d. administration. *, **, ***, **** indicate significant difference (p < 0.05, p < 0.01, p < 0.001, p < 0.0001, respectively) between i.v. and i.d. administration for each tracer size as determined by Welch ANOVA. Data are presented as mean ± SD. (E) Confocal microscope image of 30 nm PPS NP distribution within the LN 4 h post i.v. (AlexaFluor 647 labeled, green) and i.d. (Texas Red labeled, magenta) administration. Scale bar represents 100 μm. In A–D, n = 5 mice per group and results are representative of two independent experiments.