Lin− cells were first stained with phycoerythrin-indotricarbocyanine to ensure any residual Lin+ cells could be gated out, then were stained with various combinations of monoclonal
antibodies to CD117 (c-Kit; ACK-2; conjugated to fluorescein isothiocyanate or allophycocyanin), CD43 (S7; conjugated to biotin), CD115 (M-CSF receptor; Neratinib AFS98; conjugated to biotin), and CD16/32 (2.4G2; conjugated to allophycocyanin). Streptavidin-phycoerythrin was then used to stain biotin-binding cells. At the end of the culture period, a fixed number of latex beads was then added to each culture to aid in the quantification of DCs. Cells were stained with anti-CD11c (N418), anti-Sirpα (P84), anti-CD45RA (14.8), and antibody to MHCII (M5/114), with propidium iodide (1 μg/mL) added to the final wash to stain dead cells. DC progeny were then counted by flow cytometry, with gating on viable CD11c+ MHCII+ cells and
CD45RAhiSirpαlo DCs (pDCs), CD45RA−Sirp-αhi DCs (CD8−cDC–equivalent cells) and CD45RA− Sirp-αlo DCs (CD8+ cDC-equivalent cells). Flow Jo software was used to analyze the data. BM-derived DCs were stimulated with 0.5 mg/mL PMA for 10 min, and then incubated with 2 mM redox sensitive probe, 5- (and 6-) chloromethyl-29,79 dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) for 20 min at 37°C. Control cells were treated redox sensitive probe and CM-H2DCFDA Selleck Gefitinib only. The intracellular ROS level of defined populations was measured by the oxidation of the probe (detected by the increase of FITC fluorescence). The true level of intracellular ROS was estimated by subtracting the background mean fluorescence intensity (MFI) of the negative control from the MFI values of fluorescent samples as RANTES measured by flow cytometric analysis. Purified BM-DCs were resuspended at 0.5 × 106/mL in fresh
media in the presence or absence of a single TLR agonist, CpG ODN 1826 (1 μM) (Coley Pharmaceutical, Ottawa, Canada), and cultured for 20 h before supernatants were collected and analyzed for IL-12p70, IL-10, and TNF-α by ELISA according to the manufacturer’s instructions (BD Biosciences). (35S) met/cys labeling of newly synthesized proteins, immunoprecipitations, and autoradiography were conducted as previously described [47]. Normalization of (35S)met/cys incorporation was conducted by pipetting 7 μL of cell lysate on filter paper, washing the paper four times in 1% TCA (Sigma-Aldrich), and counting the amount of radioactivity precipitated on the paper in a scintillation counter (HIDEX 300SL, Finland). The amount of cell lysate used for immunoprecipitation for targeted proteins was then adjusted accordingly to ensure equal amounts of radiolabeled materials from each sample. Mice were injected i.v. with 2 × 10 4 cells L. monocytogenes.