Apc anti mouse cd8 antibody

Mouse spleen cells were harvested and divided into three parts. The first part of the spleen cells were treated with 100μL of 20μg/mL FITC anti-mouse CD3 antibody (BioLegend, San Diego, USA), 100μL of12.5μg/mL Percp anti-mouse CD4 antibody (BioLegend) and 100μL of 12.5μg/mL APC anti-mouse CD8 antibody (BioLegend) at 25°C for 30 min, washed, and followed by incubation with 100μL of 10μg/mL FITC-conjugated goat anti-rat IgG (BioLegend) and incubated at 25°C for 30 min in the dark. The second part of the spleen cells were treated with 100μL of 20μg/mL FITC anti-mouse CD3 antibody (BioLegend), 100μL of 12.5μg/mL Percp anti-mouse CD4 antibody (BioLegend),100μL of 25μg/mL APC anti-mouse CD25 antibody (BioLegend) and 100μL of 20 μg/mL PE anti- mouse FOXP3 (BioLegend) using the same protocol as above. The third part of spleen cells were treated with 100μL of 20μg/mL FITC anti-mouse CD19 antibody (BioLegend) and 100μL of 50μg/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, then quantified by flow cytometry (BD Calibur™).

The perfused mouse brains were rinsed in Hank’s buffer solution to remove the surface foreign matter, and the brain tissues were cut to the size of rice grains with surgical scissors and added with 1 mg mL⁻¹ collagenase IV (Sigma-Aldrich) and 50 U mL⁻¹ DNase I (Sigma-Aldrich, 15 kU) in 1640 medium in 5 mL of tissue digest, placed on a 37 °C shaker, and fully digested for 1 h. The entire digested tissue homogenate was poured into a 70 µm nylon filter, and the tissue residue was filtered by grinding the tissue with the flat end of a syringe, the above cell suspension centrifuged (200 × g, 5 min) to remove the supernatant. The cells were blown well by adding staining buffer. CD3⁺CD8⁺ T cells were stained with FITC-anti-mouse CD3 antibody (dilution of 1:50, catalog number: 100203, clone: 17A2, Lot: N418 Biolegend) and APC anti-mouse CD8 antibody (dilution of 1:80, catalog number: 100711, clone: 53-6.7, Lot: B329662, Biolegend). Flow cytometry data was obtained from flow cytometry (BD Accuri C6 Plus flow cytometer) and analyzed with CytExpert (2.3) software and FlowJo (Version 10).

Mouse blood cells were harvested and divided into three parts. First, spleen cells were treated with 100 μL of 20 μg/mL FITC anti-mouse CD3 antibody (100306, BioLegend, San Diego, USA), 100 μL of 12.5 μg/mL Percp anti-mouse CD4 antibody (100407, BioLegend), and 100 μL of 12.5 μg/mL APC anti-mouse CD8 antibody (100732, BioLegend) at 25°C for 30 min, washed, then incubated with 100 μL of 10 μg/mL FITC goat anti-mouse IgG antibody (405305, BioLegend) at 25°C for 30 min in the dark. Second, blood cells were treated with 100 μL of 12.5 μg/mL Percp anti-mouse CD4 antibody (BioLegend), 100 μL of 25 μg/mL APC anti-mouse CD25 antibody (101910, BioLegend), and 100 μL of 12.5 μg/mL PE anti-mouse Foxp3 antibody (320008, BioLegend) via the same protocol described above. Third, blood cells were treated with 100 μL of 20 μg/mL FITC anti-mouse CD19 antibody (115506, BioLegend) using the same protocol described above. After incubation, cells were washed and resuspended in 0.5 mL of PBS/2% paraformaldehyde and quantified by flow cytometry (BD Calibur^TM).