Alexa fluor 647 antimouse cd86

For the analysis of DC maturation, TDLNs were collected 5 days post different treatments. The collected lymph nodes were ground and filtered through 100 µm cell strainers to prepare single cell suspensions. Then, the cells were stained with Alexa Fluor 488 antimouse CD11c, Alexa Fluor 647 antimouse CD86, and PE antimouse CD80 antibodies (BioLegend, USA) and then detected by flow cytometry (BD Calibur). For detecting the infiltration of CD8⁺ T cells, tumors of mice in different groups were harvested at observe endpoint (Day 16) and cut into small pieces. Then, samples were digested in 2 mL RPMI containing 500 µg mL⁻¹ collagenase IV (Invitrogen), 100 µg mL⁻¹ hyaluronidase, and 20 U mL⁻¹ DNase (Macklin) at 37 °C for 30 min and single cell suspensions were prepared after filtered through a 300‐mesh cell screen. Cells were next stained with PE antimouse CD3 and APC antimouse CD8a (BioLegend, USA) for flow cytometry analysis. Tumor tissues were also collected for immunohistochemical staining to further evaluate tumor infiltration. Furthermore, IFN‐γ within tumors was detected by ELISA kits (Dakewe Biotech Co., Ltd.).

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).