Transcription factor buffer set

All cell suspension samples were adjusted at 10⁶ cells per 50 μl before running the flow cytometry. Cells were then incubated for 10 min at 4°C with anti-mouse CD16/32 (Biolegend, USA) for blocking Fc receptors. Surface molecules of DCs were stained for 30 min at 4°C with the following antibodies: anti-mouse CD11c Percp-cyanine 5.5 (eBioscience, USA), anti-mouse lineage brilliant violet 421, anti-mouse B220-APC-Cy7, anti-mouse CD40-APC, anti-mouse CD80-PE, anti-mouse CD86-FITC, and anti-mouse MHC-Ⅱ PE-cyanine 7 (Biolegend, USA). Treg cells were stained with surface markers: anti-mouse CD4-FITC (Biolegend, USA) and anti-mouse CD25-APC (eBioscience, USA). The intranuclear Foxp3 of Treg cells was stained with anti-mouse Foxp3-PE (eBioscience, USA) following cell fixation and permeabilization with the transcription factor buffer set (BioLegend, USA). Cellular fluorescence was assessed with FACS Canto II (BD Biosciences, USA) and data were analyzed with FlowJo software (version 10.2, TreeStar, USA) [29 (link)].

PBMCs were isolated from AIH patients and healthy volunteers as mentioned above. In addition, single-cell suspensions were obtained by mechanical disruption of mouse spleens through 70-μm cell strainers. Erythrocytes were lysed using lysis buffer. Then, the freshly collected cells were resuspended in 100 μl of PBS and incubated with anti-CD3, anti-CD4, and anti-CD8a antibodies (BioLegend, San Diego, CA, United States ) at 4°C for 30 min. To detect Treg subpopulations, intracellular staining was performed using a transcription factor buffer set (BioLegend, San Diego, CA, United States ). After staining with anti-CD4 and anti-CD25 antibodies, the cells were fixed, permeabilized, and incubated with an anti-FOXP3 antibody (BioLegend, San Diego, CA, United States ). The frequencies of CD4+ CD25+ FOXP3+ Tregs, CD3+ CD4+ T cells, and CD3+ CD8a+ T cells were examined using a flow cytometer (Beckman Coulter, Brea, CA, United States).

Sections of the spleen, axillary lymph nodes, and tumors were prepared as single cell suspensions. The following flow cytometry antibodies were use: anti-mouse CD3 PE-Cyanine5 (15-0031-81; eBioscience), anti-mouse CD4 PE(12-0041-81; eBioscience), anti-mouse CD 8 FITC (12-0081-81; eBioscience), anti-mouse LY-6C APC (17-5932-80; eBioscience), anti-mouse LY-6G FITC (11-9886-80; eBioscience), anti-mouse F4/80 PE (565410; BD Pharmingen), anti-mouse CD11b APC-Cy7 (561039; BD Pharmingen), anti-mouse CD4 PE-Cyanine5 (15-0041-81; eBioscience), anti-mouse CD25 PE (12-0251-81; eBioscience), anti-mouse Foxp3 Alexa Fluor^® 488 (126405; Biolegend). Intracellular staining was performed after conducting fixation and permeabilization using Transcription Factor Buffer Set (424401; Biolegend). Cells were incubated on ice for 30 min before analyzed the samples using a FACS Canto II cytometer. The resulting data were processed using FlowJo (version 10.0.7) software.

Stimulated cells were seeded in a 96-well v-shaped staining plate (Sarstedt) at a concentration of 1 × 10⁶ cells/well. A FACS panel was created to characterize the polarized macrophages. Cells were first stained with the LIVE/DEAD Fixable Dead Cell Stain Kit-Aqua (Life Technologies) according to the manufacturer's instructions. Blocking of the cell surface Fc receptor was performed with 10% human serum. Staining of the cell surface markers was performed using the following antibodies from BioLegend: CD80, CD209, CD206, HLA-DR, and CD11b. After surface staining, cells were washed and fixed/permeabilized with the transcription factor buffer set (BioLegend) according to the manufacturer's instructions. Intracellular blocking was performed with 10% human serum. The cells were then stained for intracellular CD68 using an antibody from BD Biosciences. Stained cells were washed, resuspended in FACS wash buffer and acquired using a FACS verse instrument and FACS Suite software (BD Biosciences). Data analysis was performed using Flow Jo software.