Anti ly6c apc

IMQ was purchased from InvivoGen (SanDiego, CA, USA). 3-MA was purchased from Sigma-Aldrich (St. Louis, MO, USA). For western blot analysis, anti-β-actin, anti-LC3B, anti-Beclin-1 and anti-Atg5-12 antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-phospho-Erk1/2, anti-Erk1/2, anti-phospho-p38, anti-p38 and anti-phospho-IκBα were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-phospho-NF-κB p65, anti-NF-κB p65 and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The other reagent sources were as follows: diphenyleneiodonium chloride (DPI) (Calbiochem, USA), apocynin (Sigma-Aldrich, USA), and DCFH-DA (Calbiochem, USA). Anti-CD4-FITC, anti-CD8-FITC, anti-CD11b-FITC, anti-FOXP3-Alexa 488 (126406), anti-Ly6C-APC, anti-CD45 (103124), anti-Ly6G-PE/Cy7 (15-5931-82), anti-TNFα-APC (17-7321-82), anti-IFN-γ-APC (17-7311-82), anti-Gr-1-Alexa 488 (108417) and anti-CD25-Alexa 647 (102020) antibodies were purchased from eBioscience (San Diego, CA, USA). For immunostaining, an anti-LC3 antibody was purchased from MBL International Corporation (Nagoya, Japan), and a FITC-conjugated anti-rabbit secondary antibody was purchased from Jackson Immuno Research (West Grove, PA, USA).

We collected 100-µl aliquots of tail blood into EDTA-containing tubes. Cells in some tubes were Fc-blocked with 1 µg of mouse IgG (105 cells) for 15 min at room temperature without excess washing. After blocking, cell suspensions were then labeled with several monocyte-surface markers (anti-CD115–PE, eBioscience, catalog #17-1152; and anti-Ly-6C–APC, eBioscience, catalog #12-5932) and anti-CCR2–FITC (B&D, catalog: FAB5538F) or anti-CX3CR1-FITC (catalog #FAB5825G, R&D Systems, Minneapolis, MN, USA) at 4°C for 30 min. After cells were lysed by the addition of 4 ml Whole Blood Lysing Reagent (catalog #FC002, R&D Systems), the cells were washed twice with ice-cold PBS followed by centrifugation at 300× g for 5 min. The pellets were resuspended by adding 150 µl PBS. Controls included cells incubated with fluorochrome-conjugated unspecific isotype Ab as needed. Cell-surface molecule expression measurement and cell sorting were performed on a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Acquired fluorescence-activated cell sorting (FACS) data files were analyzed using the flow cytometry CellQuest software (BD Biosciences). Blood CD115+/Ly-6high cells defined as monocytes were further analyzed for CX3CR1 and CCR2 expression.

Whole-blood samples (15 μL) were washed with cold PBS before incubating for 20 min on ice with directly conjugated antibodies: anti-Ly6C-APC (eBioscience, Cheshire, UK), anti-CD11b (eBioscience, Cheshire, UK) and anti-Ly6G (Gr1)-FITC (eBioscience, Cheshire, UK). Monocytes were gated according to CD11b expression and side scatter and then further separated by Gr1 and Ly6C staining. Two monocyte subsets were identified as SSC^lowCD11b⁺Gr1 ⁺ Ly6C^high and SSC^lowCD11b⁺Gr1^-Ly6C^low monocytes, whereas neutrophils were identified as SSC^highCD11b⁺Gr1^highLy6C^inte (Figure 3A and B) using FACSCalibur. Combination of anti-CD11b, anti-Gr1 and anti-Tie2-PE (eBioscience, Cheshire, UK) were used to stain cells to analyse Tie2-positive monocyte population.