Hamster anti mouse cd11c

The FACS analysis procedure was adapted from previous work.¹⁰ Two days after the last intraarticular particle injection, the mice were given P-Dex-Alexa 488 (5 mg/mouse) via tail vein injection. At necropsy (24 hr post injection), the left femurs were isolated and minced aseptically. The tissues were further digested with type IA collagenase (1 mg/mL, Sigma-Aldrich) at 37 °C for 30 min twice. After passing through a 70 μm cell strainer, ACK Lysing Buffer (Quality Biological, Gaithersburg, MD) was then used to remove the red blood cells. After centrifugation (1200 rpm, 5 min), a single cell suspension (1×10⁶ cells/50 μL) was obtained. For FACS evaluation of dendritic cells, macrophage, monocytes and fibroblast cells, the samples were incubated with the following antibodies: hamster anti-mouse CD11c (BD Biosciences, Pharmingen), Allophycocyanin (APC)-labeled rat anti-mouse F4/80 (eBioscience, San Diego, CA), APC-labeled rat anti-mouse Ly-6G (Gr-1, Gr1) (eBioscience, San Diego, CA) and Alexa Fluro^® 647 labeled rabbit anti-mouse P4HB (Abcam, Cambridge, MA), respectively for 30 minutes on ice. Cells incubated with hamster anti-mouse CD11c were further treated with Alexa Fluro^® 647-labeled goat anti-hamster secondary antibody for another 30 minutes on ice. All the cells were then fixed in FACS fixation buffer and stored at 4 °C prior to analyses on a BD^™ LSR II flow cytometer.

Tumor samples were collected and progressively frozen in cold 2-methylbutane solution. Sections (6μm) were fixed in 100% cold acetone, blocked with 8% normal goat serum for 2 hours, and incubated with the appropriate primary antibodies (rat anti-mouse CD11b, Hamster anti-mouse CD11c, rat anti-mouse CD8a or rat anti-mouse CD11b, BD Biosciences) for 2 hours at room temperature. Sections were washed 3 times with PBS and incubated with goat anti-rat secondary antibodies coupled to Alexa488 or 597 for CD11b, CD8 and CD4 studies or with Cy5-goat anti-Hamster for CD11c studies. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). The slides were washed and mounted in DAKO fluorescent mounting medium. The detection of apoptotic cells was performed using a TUNEL-assay (ApoTag Fluorescein In Situ Apoptosis Detection Kit, Promega) in accordance with the manufacturer’s instructions. Immunofluorescence images were collected on a Nikon microscope (Eclipse TE2000-U) and pixels of fluorescence or area of staining was quantified using MetaMorph Software.