Anti fcγiii 2 receptor antibody

After 24 or 48 hr culture, scaffolds were removed from the culture medium and JAWSII cells grown inside or on the surface of the scaffolds were retrieved by PBS-based cell dissociation buffer (Invitrogen). After centrifugation, cells were suspended in 200 μL FACS buffer (DPBS containing 2% FBS) at concentration 1 × 10⁶ cells/ml. Nonspecific binding was blocked by incubating with anti Fcγ III/II receptor antibody (1:500) (BD Biosciences) at 4 °C for 5 min. Cells were then incubated with CD86 (BioLegend, San Diego, CA, APC-conjugated, 1:1000), CD80 (BioLegend, PerCP-Cy5.5-conjugated, 1:1000), or MHCII (BioLegend, PE-conjugated, 1:1000) antibodies on ice in the dark for 30–40 min. Cells were washed twice with PBS and re-suspended in 200 μL FACS buffer with 0.2 μg/ml propidium iodide (Invitrogen). To determine surface antigen expression levels, 10,000 events per sample gated on viable propidium iodide-negative single cells were acquired on BD FACSCantoII flow cyotometer (BD Biosciences). Cells were gated according to positive APC, PerCP-Cy5.5, or PE using unstained controls, and the percentage of cells staining positive for each surface antigen was recorded and analyzed using FlowJo software (TreeStar).