Alexa fluor 488 anti mouse cd80

For immunophenotyping macrophages, control and anti-CD47 mAb-treated mice were killed, and subcutaneous tumors were dissociated to single cells and stained with APC/Cy7 anti-mouse LY-6G/Ly-6C (Gr-1) (BioLegend), PE/Cy7 anti-mouse CD45 (BD Bioscience), PerCP/Cy5.5 anti-mouse/human Cd11b (Biolegend), APC anti-mouse F4/80 (eBioscience), Brilliant Violet 785™ anti-mouse I-A/I-E (MHC-II) (BioLegend), Brilliant Violet 605™ anti-mouse CD86 (BioLegend), PE anti-mouse CD206 (BioLegend), PE/Cy7 anti-mouse CD124 (IL-4R alpha) (BioLegend), and Alexa Fluor® 488 anti-mouse CD80 (BioLegend), all according to the manufacturers’ specifications. Endogenous mouse FcγII and FcγII receptors were blocked using 3% BSA containing Mouse BD Fc Block^TM (1 μg). Flow cytometric analysis was performed on a BD FACS ARIA II (BD) flow cytometer. Data analysis was performed using FlowJo Version 9.6.4 (Tree Star, Ashland, OR, USA). Live singlets were gated using FSC-W/FSC-H. Gates were drawn using Fluorescent Minus One control tubes.

Aire⁺ cells were grown to semi-confluency or confluency in RepCell culture dish (CellSeed). Aire⁺ cells were detached by gentle pipetting at 20°C. The detached cells were washed once with cold PBS and fixed with 4% paraformaldehyde in PBS (pH 7.4) for 15 min on ice. Then, Aire⁺ cells were stained with fluorescent antibodies specific for each of IS components CD40 (Alexa Fluor 647 anti-mouse CD40, BioLegend), MHC-II (Alexa Fluor 488 anti-mouse I-A/I-E, BioLegend), CD80 (Alexa Fluor 488 anti-mouse CD80, BioLegend), CD86 (Alexa Fluor 647 anti-mouse CD86, BioLegend), and control IgG. After washing with cold PBS, surface fluorescence of the stained Aire⁺ cells (5,000 cells each) was measured by a flow cytometry system Guava (Millipore).