40
Uptake of Fluorescent Labeled Proteins or Particles
To assess uptake of particulate antigen, standard techniques utilizing fluorescent labeled dextran, ovalbumin, and BSA were utilized. Dendritic cells were suspended in
1 mL of RPMI-C with 5 mM HEPES, and 1 mg of Rhodamine Green-Dextran, DQOvalbumin, or DQ-BSA (Molecular Probes, Eugene, OR). Cells were incubated for 6 hours at either 4o or 370, and then analyzed for fluorescence. Both DQ-OVA and DQ-BSA are self-quenched BODIPY dyes that must be cleaved in order for fluorescence to be seen. Additionally, both the Rhodamine green and BODIPY dyes are pH insensitive. Thus, no signal is lost due to the pH of the lysosomal compartment. ELISA for Cytokine in Culture Supernatants
Cytokine concentrations in culture supernatant were analyzed using products from Pharmingen (San Diego, CA) unless specified otherwise. The following antibody pairs were utilized for cytokine detection, listed as capture and detection respectively; IL-2 (JES6-1A12 & JES6-5H4), IL-4 (BVD4-1D 11 & BVD6-24G2), IFN-y (R4-6A2 & XMGl.2), IL-10 (JES5-2A5 & SXC-1), IL-12p40 (C15.6 & C17.8), IL-12p70 (9A5 & C 17.8), A streptavidin-HRP secondary was added, followed by addition of a TMB substrate. The reaction was stopped using IN H2SO4 and read at 450 nm on a 3550-UV microplate reader (Bio-Rad, Hercules, CA).
Results
Identification of Protective Dendritic Cell Populations
Revisiting an Old Friend
In the early 1990's, our lab performed experiments involving adoptive transfer of dendritic cell populations to juvenile NOD mice. These dendritic cells migrated from the foot pad to the popliteal lymph node where they stimulated cellular proliferation and