Fitc conjugated anti cd11c

The cells were washed twice and cells (1 × 10⁶) were suspended in 50 μl PBS supplemented with 1% FBS and stained for 20 min at 4 °C with directly conjugated fluorescent antibodies (1 : 500). Antibodies were as follows: eFluor450-conjugated anti-CD11b (eBioscience); FITC-conjugated anti-Gr-1 (BD Biosciences, Franklin Lakes, NJ, USA); APC-conjugated anti-Gr-1 (eBioscience); FITC-conjugated anti-CD11c (eBioscience). Stained cells were analysed with a FACS Canto flow cytometer using FACS Diva software (BD Biosciences), and the data were analysed with FlowJo software (TreeStar, Ashland, OR, USA). Cells were sorted with a FACS Aria flow cytometer (BD Biosciences) and washed twice and suspended in PBS. Cells (2 × 10⁵) were resuspended in 100 μl PBS and centrifuged onto microscope slides using a Cytospin-4 (Thermo Scientific, Waltham, MA, USA). Slides were then stained by May-Grunwald Giemsa according to a standard protocol.

Annexin V-fluorescein isothiocyanate (FITC) staining was used to analyze the apoptosis of osteosarcoma cells, according to previously published protocols.15 (link) The purity and phenotype markers of peritoneal macrophages were analyzed by FACS with phycoerythrin (PE)-conjugated anti-F4/80, FITC-conjugated anti-CD11c, and PE-conjugated anti-mouse CD16/32 antibodies. The information of the antibodies used is as follows: PE-conjugated anti-F4/80 is a monoclonal antibody (BM8) and was purchased from eBioscience (cat. no. 12-4801-80). FITC-conjugated anti-CD11c is a monoclonal antibody (N418) and was purchased from eBioscience (cat. no. 11–0114-82). PE-conjugated anti-mouse CD16/32 antibody was purchased from BioLegend corporation (cat. no. 101307). Briefly, 1 × 10⁶ Hepa1-6 or H22 cells were washed twice in cold PBS. The PE-conjugated or FITC-conjugated antibodies were added to a final concentration of 100 ng/mL each. The antibodies were incubated for 20 min at 4°C in the dark, prior to flow cytometry analysis (FACScan; Belgium).