Pe anti mouse foxp3 antibody

Cells in the SVFs were suspended in the staining buffer (PBS containing 0.5% BSA and 2 Mm ethylenediaminetetraacetic acid) and then incubated with CD11b (BioLegend, clone: M1/70) and CD11c (BioLegend, clone: N418) antibodies or the control isotypes at 4 °C. Thirty minutes later, cells were then washed twice and resuspended in the staining buffer. After incubation with 7-amino-actinomycin D (BioLegend), the cells were analyzed by Attune NxT Flow Cytometer (ThermoFisher). The data analysis was performed by using FlowJo (Tree Star, Ashland, OR). Cells with double positive expression of CD11b and CD11c were identified as M1 macrophages. For intracellular FoxP3 staining, PE/Cyanine7 anti-mouse CD4 (BioLegend, # 100,528)-stained cells were resuspended in 1 ml of the True-Nuclear™ 1X Fix Concentrate to each tube, vortex and incubate at room temperature in the dark for 60 min, followed by adding 2 mL of the True-Nuclear™ 1X Perm Buffer (BioLegend, # 424,401) to each tube and then centrifuge tubes at 2100 rpm at room temperature for 8 min, and discard the supernatant. Tubes were decanted, blotted, washed twice with True-Nuclear™ 1X Perm Buffer. Resuspend the cell pellet in 100 µl of the True-Nuclear™ 1X Perm Buffer. For detection of intracellular antigen, add 1 μl of PE–anti-mouse FoxP3 antibody (BioLegend, # 126,404) to each tube and incubate in the dark at room temperature for 60 min.

To measure expressions of surface molecules, cells were stained with FITC anti-mouse major histocompatibility complex (MHC)-II antibody (1:200; Biolegend, 107606), BV510 anti-mouse CD3 antibody (1:200; Biolegend, 100234), FITC anti-mouse CD4 antibody (1:200; Biolegend, 100405) or APC anti-mouse CD25 antibody (1:200; Biolegend, 101909) in FACS buffer for 40 min. For intracellular staining, cells were permeabilized, followed by fixation using the Foxp3 staining buffer kit prior to staining with PE anti-mouse Foxp3 antibody (1:100; Biolegend, 320007), BV421 anti-mouse Foxp3 antibody (1:100; Biolegend, 126419), or PE-conjugated S100A9 antibody (1:100; Cell Signaling Technology, 93941). Flow cytometry analyses were performed on a LSR II instrument and raw data were retrieved. Results were analyzed by use of FlowJo software version 10.0.

Mouse spleen cells were harvested and divided into three parts. The first portion of the spleen cells was treated with 100 μL of 20 μg/mL FITC anti-mouse CD3 antibody (BioLegend, San Diego, CA, USA), 100 μL of 12.5 μg/mL PerCP anti-mouse CD4 antibody (BioLegend), 100 μL of 25 μg/mL APC anti-mouse CD25 antibody (BioLegend), and 100 μL of 20 μg/mL PE anti-mouse Foxp3 antibody (BioLegend) at 25 °C for 30 min, washed, and then incubated with 100 μL of 10 μg/mL FITC-conjugated goat anti-rat IgG (BioLegend) at 25 °C for 30 min in the dark. The second portion of the spleen cells was treated with 100 μL of 20 μg/mL FITC anti-mouse CD3 antibody (BioLegend), 100 μL of 12.5 μg/mL APC anti-mouse CD8 antibody (BioLegend), and 100 μL of 12.5 μg/mL PE anti-mouse CD28 antibody (BioLegend) using the same protocol as above. The third portion of spleen cells was treated with 100 μL of 20 μg/mL FITC anti-mouse CD19 antibody (BioLegend) and 100 μL of 50 μg/mL PE anti-mouse CD80 antibody (BioLegend) using the same protocol as above. After incubation, the cells were washed and resuspended in 0.5 mL of PBS/2% paraformaldehyde, and then quantified using flow cytometry (BD Calibur™, San Jose, CA, USA).