Quantificationof NP LN accumulation

To determine the amount of NP accumulated in draining LN, either fluorescence quantification of fluorescently labelled NP^5,55 or a modified assay measuring PEG using Iodine and barium chloride, which form a barium-iodide complex of the glycol, was performed.^58,59 For the fluorescent quantifcaiton of percent of injection, LN homogenate was taken and read at 655/675 nm, and the concentration of AlexaFluor647-NP was determined using a standard curve of AlexaFluor647-NP that was allowed to react at 37°C for 24 hr. For the Iodine PEG assay centrifuged LN homogenate, BaCl solution in 1N HCL, and KI solution supplemented were reacted. The absorbance was measured at 535 nm, and the concentration of NP was determined using a standard curve of NP diluted in 1× PBS.