2 μl of a 1% solids NP solution, and incubated at room temperature for 30 min. To determine binding of Ag to the NP,
the Z potential of NP was tested before and after protein adsorption, since proteins modify the NP surface charge. Adsorption was also tested by Bradford assay, and for gp140 a specific anti-gp140 ELISA was performed. Because the NP are made of wax material, it was not possible to spin down the NP for further testing of unbound Ag present in the supernatant, therefore a different protocol had to be used. After incubation of NP with Ag, the mix was spun at 4000 × g for 10 min using a 1,000,000 MW cut-off Vivaspin filter (Sartorius Stedim Biotech, Goettingen, Germany). Antigen alone was spun in parallel to control for the amount of Ag retained in the filter. BKM120 After centrifugation, the www.selleckchem.com/products/CAL-101.html NP were retained in the filter and the amount of Ag present in the filtrate was then tested by Bradford and ELISA assays. To determine the amount of Ag adsorbed to NP, the amount of Ag detected in the colorimetric assays was calculated as a percentage of the amount of Ag alone recovered
after filtration. Co-adsorption of Ag with the TLR-9 ligand CpGB or PolyI:C was performed using the YC-Brij700-chitosan NP, which are positively charged. CpGB (Eurofins MWG Operon, Ebersberg, Germany) and Poly (I:C) (Invivogen, San Diego, CA) was added to the NP-Ag complex at 4.25 μg/ml final concentration and incubated for an additional 30 min. CpGB and Poly (I:C) binding was assessed using the PicoGreen dsDNA and RiboGreen dsRNA quantitation reagents (Invitrogen Ltd., Paisley, UK). Buffy coats obtained from healthy volunteers were used for separation of mononuclear cells (MNC) by density gradient centrifugation using ficoll-hypaque (Histopaque, Sigma). Monocytes were separated from non-adherent cells by adherence to plastic using complete medium
(CM: RPMI-1640 plus 10 mM HEPES, 2 mM l-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, all from Sigma) supplemented with 0.5% AB pooled human serum (PHS, Dynal Biotech, Ullernchausseen, Norway). Adherent cells were then cultured for 4 days with 15 ml CM supplemented with 5% PHS that contained 25 ng/ml GM-CSF and 30 ng/ml Resminostat IL-4 (R&D Systems, Inc., Minneapolis, MN). Complete medium with cytokines was replaced and after 3 days the cells were recovered and tested for cell morphology by optical microscopy, and for DC phenotype by immunofluorescence and flow cytometry. Cells were placed in glass-bottom culture dishes (MatTek, Co., Ashland, MA) in CM plus GM-CSF and IL-4. The microscope and stage were enclosed within a heated (37 °C) chamber (Solent Scientific, UK) and cells were cultured in 5% CO2 in air. Images were captured using an Olympus IX71 inverted fluorescence microscope equipped with a Hamamatsu C4742-95 digital camera, using a 20× objective with an additional 1.6× adaptor. Captured images were analyzed using Image Pro Plus software (Media Cybernetics, USA).
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