Anti cd11c pe

The SVF cells and splenocytes (2.5 × 10⁵ cells/sample) were incubated with Fc-blocker (anti-CD16/CD32; eBioscience, San Diego, CA) for 20 min and then stained with combinations of anti-CD11b PE-Cy7, anti-Ly-6G PE-Cy5, and anti-F4/80 PE-Cy5, anti-CD11c PE for 20 min. Heparin-treated whole blood cells (50 μL) were stained with anti-CD11b PE-Cy7, anti-Ly-6G PE-Cy5, anti-F4/80 PE-Cy5, or anti-CD11c PE (all from eBioscience). After 20-min incubation in the dark, whole blood cells were incubated with 600 μL Versa Lyse (Beckman Coulter, Tokyo, Japan) at room temperature in the dark. The SVF cells, splenocytes, and blood cells were washed with Staining buffer (BD Pharmingen). Finally, cells were resuspended in Staining buffer and analyzed using fluorescence-activated cell sorting analysis performed with Guava® EasyCyte™ 6HT flow cytometry system (Millipore, Long Beach, CA) and InCyte software (Millipore). A validation of the flow cytometric approach for the identification of total CD11b⁺ Ly-6G⁺ neutrophils is shown in Supplementary Figure S1.

The harvested BMDC (2 × 10⁶ /well) were stimulated in triplicate with LPS (1 μg/mL), alone or together with ALX at 2 or 10 μM in 6-well plates for 2 days. The cells were stained with PE-anti-CD11c, Percp-cy5.5-anti-MHC-II, FITC-anti-CD80 or FITC-anti-CD86, or isotype controls (eBioscience), washed, and analyzed by flow cytometry.
On days 24–26 post-immunization, six mice from each group were sacrificed, and their splenic mononuclear cells were isolated. The cells were stimulated with Cell Stimulation Cocktail (PMA + Ionomycin, eBioscience) for 5 h. The cells were stained with FITC-anti-CD4 (eBioscience), fixed, and permeabilized. After being washed, the cells were stained intracellularly with PE-anti-IFN-γ or PE-anti-IL-17 (eBioscience). The frequency of Th1 (CD4⁺IFN-γ⁺) and Th17 (CD4⁺Th17⁺) cells in total CD4⁺ T cells was determined by flow cytometry.
On days 15 post-immunization (the early stage of EAE), splenic mononuclear cells were isolated from the vehicle or ALX-treated EAE mice. The cells were stimulated with 20 μg/ml of MOG35-55 for 48 h and stained with PE-anti-CD11c, Percp-cy5.5-anti-MHC-II, FITC-anti-CD80 or FITC-anti-CD86, or isotype controls (eBioscience), and their expression levels were characterized by flow cytometry.

Cells were stained with propidium iodide (PI) and then evaluated for cell cycle by flow cytometry according to the manufacturer’s protocol (YEASEN, 40301ES50,). Briefly, cells seeded at 6-wells were firstly collected and wash with cold PBS, then fixed with cold 75% ethanol at 4 °C overnight. After centrifugal and washing with PBS, pellets were suspended in 500 μl of binding buffer and incubated with 10 μl of PI solution and 5 μl RNaseA at 37 °C for 30 min.
For mouse macrophages, MDA-MB-468 cells (3 × 10⁶) were subsequently injected orthotopically into nude mice. After 3 weeks, mice were sacrificed, and the tumors were dissected. Tumor tissues were minced and excised into small pieces followed by incubation in DMEM containing 1 mg/ml collagenase IV (Sigma, C4-28-100MG) and 10^–3 U/L DNase I (Invitrogen, EN0521) for 0.5-1 h. After lysed, single-cell suspensions were stained with fluorochrome-conjugated antibodies: anti-CD11c-PE (eBioscience, #12-0114), anti-F4/80-FITC (Biolegend, #123116), anti-CD206-APC (eBioscience, #17-2061) for 20 min at room temperature. After washing three times with PBS, the cells were analyzed using flow cytometry (Beckman Coulter Cytoflex) and data were analyzed by using CytExpert V2.3 and FlowJo X software version 7.6.4.