Apc cy7 conjugated anti mouse f4 80 antibody

RAW264.7 and Ana-1 macrophage cell lines were harvested and blocked with 3% BSA for 45 min, and then were stained with Alexa Flour 488 anti-mouse CD11c antibody, PerCP-Cy5.5-conjugated anti-mouse MHC Class II subunits IA-IE antibody, allophycocyanin (APC)-conjugated anti-mouse CD206 antibody or APC/Cy7-conjugated anti-mouse F4/80 antibody (Cat. #117311, #107626, #141708 and #123118, BioLegend, San Diego, CA, USA). For the analysis of macrophage marker expression in xenograft tumor tissue, tumor was minced into small pieces and incubated with 1 mg/mL collagenase Type IV and 300 U/mL DNaseI (Cat. #C5138 and #D5025, Sigma Aldrich) at 37 °C for 30 min. The tissue suspension was passed through a 70 μm cell strainer (BD Biosciences, Bedford, MA, USA), and was stained with above-mentioned fluorescein-labeled antibodies including anti-CD11c, anti-MHC II subunits IA-IE, anti-CD206 and anti-F4/80. The stained cell samples were analysed using a FACSVerse mechine (Becton Dickinson, San Diego, CA, USA). The proportion of positively-stained cells was analyzed with the FlowJo software package (Tree Star, Ashland, OR, USA).

Tumor tissue-derived cells in Buffer A (PBS with 0.5% endotoxin-free BSA and 2 mM EDTA) were counted. An aliquot of cells was pelleted and resuspended in Sorting Buffer (PBS with 0.5% endotoxin-free BSA, 2 mM EDTA and 25 mM HEPES) at 10⁷ cells/ml. Cells were incubated with Fc Block (#553142, BD Biosciences) prior to staining with conjugated antibodies for 15 min at 4 °C and then washed twice in Sorting Buffer. Cells were then resuspended in Sorting Buffer for FACS (FACSAria II or Accuri C6, BD Biosciences). The antibodies included a PerCP/Cy5.5-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) antibody (#108427, BioLegend), a PerCP/Cy5.5-conjugated anti-mouse CD90.2 antibody (#140321, BioLegend), a PerCP/Cy5.5-conjugated anti-mouse/human CD45R/B220 antibody (#103235, BioLegend), an AF700-conjugated anti-mouse CD45 antibody (#103128, BioLegend), an APC/Cy7-conjugated anti-mouse F4/80 antibody (#123118, BioLegend), a FITC-conjugated anti-mouse CD172a (Sirpα) antibody (#144006, BioLegend), a PerCP/Cy5.5-conjugated anti-human CD45 antibody (#368504, BioLegend) and an APC-conjugated anti-human CD68 antibody (#333809, BioLegend). Mouse TAMs were defined as CD90.2-CD45R-Gr1-CD45+F4/80+ cells. When macrophages were isolated from GFP-tagged MC-38 tumors, GFP+ cells were excluded. Human TAMs were defined as CD45+CD68+ cells.