Multiplex Immunophenotyping of Dendritic Cells

Following 24 h incubation on the immunoarrays, DCs were fixed with 4% paraformaldehyde (USB Corporation). Cells were then fluorescently stained either for surface markers CCR7 (Santa Cruz Biotechnology), CD86 (BD Biosciences), MHC-II (BD Biosciences), or intracellular cytokines IL-10 (BD Biosciences), or IL-12p40 (BD Biosciences) as previously reported.^{42 (link)} Intracellular staining for cytokines IL-10 and IL-12p40 was performed by first incubating seeded immunoarrays with monensin (0.7 μL mL⁻¹) for the final 8 h of culture in order to block protein secretion. The arrays were then incubated with 4% paraformaldehyde followed by 0.01% triton X-100 (Fisher Scientific). Arrays were next washed and incubated with a blocking solution consisting of 1% goat serum along with 0.01% triton X-100 in PBS for 40 min to block the background before incubation with primary antibodies. Afterward, solutions of biotinylated secondary antibodies (Invitrogen) were incubated, followed by streptavidin-cross-linked alkaline phosphatase, and lastly the precipitating fluorescent substrate, ELF97 (Invitrogen). Nuclei were stained with Hoechst 34580 dye. Immunoarrays were then mounted and imaged.