(19) were used in these PCRs. The different primer pairs were purchased from (Eurofins MWG Operon) CIITA, Fw 5′-CCCTGCGTGTGATGGATGTC-3′, Rev 5′-GTTGCCCTTAGCGTCTTCAG-3′; Li Fw 5′-GAGGCTAGAGCCATGGATGAC-3′, Rev 5′-AGATGCTTCAGATTCTCTGGG-3′; H-2Ma Fw 5′-CTACGAGATGTTGATGCGGGAAGT-3′,

Rev 5′-GTGTAGCGGTCAATCTCGTGTGTC-3′; I-a β-chain Fw 5′-GCTACTTCACCAACGGGACG-3′, Rev 5′-GCTCTTCAGGCTGGGATGCT-3′; Cat-S Fw 5′-CTTGAAGGGCAGCTGAAGCTG-3′, Rev 5′-GTAGGAAGCGTCTGCCTCTAT-3′; β-Actin Fw 5′-TGTGATGGTGGGAATGGGTCAG-3′, Rev 5′-TTTGATGTCACGCACGATTTCC-3′. Primers for CIITA detected an expected 635-bp fragment; for Li 490-bp; for H-2Ma 320-bp; for I-A β-chain 506-bp; for Cat-S 127-bp; for β-Actin AG-014699 datasheet 510-bp fragment. PCR cycling conditions were initial denaturation at 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 61°C for 30 s and extension at 72°C for 90 s. The PCR products

were stored at 4°C until use. The PCR products were analysed by electrophoresis on 2% agarose gel and ethidium bromide staining. NIH ImageJ (version 1.24t) scanning densitometer software was used to semi-quantify each band. For individual samples, the integrated intensity value of each band (sum of all the pixel intensity values in a given band) was determined, and the background was subtracted. Normalization was achieved by dividing PF-02341066 cost the corrected integrated density value of the gene in each sample by the initially corrected integrated density value of β-actin gene, which served as a control housekeeping gene to comparatively asses the corresponding sample. The ratio of the relative levels of genes (CIITA, Isoconazole li, H-2M, Ia-β chain and Cat-S) expressed in AE-pe-DCs vs. the same genes expressed in naive pe-DCs is presented by a histogram using arbitrary expression units. Immature bone marrow-derived dendritic cells (BMDCs) were generated from bone marrow precursor cells of C57BL/6 mice according to slightly modified method of (20). In brief,

bone marrow cells were harvested from the femurs and tibias of mice and plated in RPMI-1640 medium supplemented with 10% FCS, 50 μm 2-mercaptoethanol and a dose (200 U per 10 mL) of murine GM-CSF (Immunotools, Germany). A fresh culture medium containing murine GM-CSF was added every 2 days. On day 9, nonadherent cells (immature DCs) were harvested by gentle washing with PBS at 37°C. To generate BMDCs, cells were stimulated for 24 h with 1 μg/mL lipopolysaccharide (LPS; Sigma-Aldrich, Switzerland) and seeded to a 96-well-round bottom microtiter plate at a density of 106 cells per well. The cells were then incubated during 2 h at 37°C in 100 μL PBS containing E/S products (5 μg protein per mL), V/F (50 μg protein per mL) or with medium containing 50 μg BSA only (as a mock control), respectively. Then, plates were centrifuged, supernatant was removed and BMDCs were processed for membrane protein extraction.