Anti mhcii

Fluorescein isothiocyanate (FITC)-conjugated Annexin V/propidium iodide (PI) Kit, anti-CD80, anti-CD86, and phycoerythrin-conjugated anti-MHC-I and anti-MHC-II were purchased from BD Biosciences (San Diego, CA, USA). Lipopolysaccharide (LPS) from Escherichia coli O111:B4 was purchased from InvivoGen (San Diego, CA, USA). Anti-phosphorylated extracellular signal-regulated kinase (ERK)-1/2 monoclonal antibody (Ab), anti-phosphorylated p38 monoclonal Ab, anti-phosphorylated c-Jun N-terminal kinase (JNK) monoclonal Ab, anti-phosphorylated inhibitor of κB (IκB)-α monoclonal Ab, anti-IκB-α monoclonal Ab, anti-nuclear factor (NF)-κB (p65) polyclonal Ab, anti-lamin B polyclonal Ab, anti-Toll-interacting protein (Tollip) monoclonal Ab, anti-suppressor of cytokine signaling (SOCS) 1 polyclonal Ab, anti-interleukin (IL)-1 receptor-associated kinase (IRAK)-M monoclonal Ab, anti-TLR4 polyclonal Ab, anti-cyclooxygenase (COX)-2 polyclonal Ab, anti- inducible nitric oxide (NO) synthase (iNOS) polyclonal Ab, and anti-β-Actin monoclonal Ab were obtained from Cell Signaling Technology (Danvers, MA, USA). Goat anti-rabbit-IgG-horseradish peroxidase (HRP) Ab and goat anti-mouse-IgG-HRP Ab were purchased from Calbiochem (Merck KGaA, Darmstadt, Germany), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

pDCs were harvested after activation with IMQ and CpG for 48 hours, and stained with the following mAbs: anti-CD11c, anti-CD11b, anti-B220, anti-CD80, anti-CD86, anti-MHC II (BD Pharmingen, USA), anti-TRAIL, and anti-Granzyme B (eBioscience, USA). Mice were injected i.t. with resting or activated pDCs, and sacrificed on day 2 or 5. Single-cell suspensions were prepared from tumor tissues. After enzymatic digestion for 30 minutes at 37°C with type IA collagenase (1 mg/mL) and DNase (0.1 mg/mL), red blood cells were lysed with Pharmlyse Buffer (BD Biosciences, USA); and stained with the following mAbs: anti-CD45, anti-NK, anti-CD3, anti-CD8, anti-TRAIL, anti-NKG2D, anti-NKG2A, and CD107 (BD Pharmingen, USA). Intracellular staining was performed using a cell permeabilization kit (Fix & Perm; An Der Grub). After incubation with the respective Abs for 20 minutes at 4°C, cells were washed twice and subjected to flow cytometric analysis. FACS plots depict the mean fluorescence intensity (MFI) values of Ab staining after subtraction of the MFI of the respective isotype.

Anti-CD4, anti-CD8, anti-CD11c, anti-CD11b, anti-MHC-II, anti-CD80, anti-CD86, anti-CD335, anti-CD3, anti-IFN-γ (all BD Biosciences, Heidelberg, Germany), anti-CD62L, anti-TNF-α, anti-Foxp3 (all eBioscience, Frankfurt, Germany), anti-CD160, anti-CD138, and anti-IL-10 (Biolegend, London, UK) were used as fluorescein isothiocyanate, pacific blue, phycoerythrin (PE), BD Horizon V450, allophycocyanin, AlexaFlour647, PE-cyanin 7, or peridinin-chlorophyll protein conjugates. Dead cells were identified by staining with the fixable viability dye eFlour 780 (eBioscience, Frankfurt, Germany). Intracelluar staining for Foxp3 was performed with the Foxp3 staining kit (eBioscience, Frankfurt, Germany) according to the manufacturer’s recommendations. Cytokine production from freshly isolated splenocytes was measured by stimulating cells with 10 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, München, Germany) and 100 µg/ml ionomycin (Sigma-Aldrich, München, Germany) for 4 or 6 h (IL-10), respectively, in the presence of 5 µg/ml Brefeldin A (for IFN-γ, TNF-α staining) and 5 µg/ml Monensin (for IL-10 staining), treating with 2% paraformaldehyde and 0.1% NP40, and staining with the respective antibody cocktail. Flow cytometric expression analyses were performed with an LSR II instrument using DIVA software (BD Biosciences, Heidelberg, Germany).

RAW264.7 cells were harvested and preincubated with 0.5% BSA diluted in PBS for 30 min. The cells were then probed with fluorescence-conjugated anti-CD80, anti-CD86, anti-MHC-I, and anti-MHC-II (BD Biosciences) antibodies at 4 °C for 30 min. All Abs were diluted 100-fold before use. After washing with PBS, the expression of the corresponding molecules was measured by flow cytometry. The data were analyzed using FlowJo software.