Anti cd4 apc

Antibodies including APC-anti-CD4 (Invitrogen, Clone: RM4-5, Catalog: 17-0043-82) or PE-Cy7-anti-CD4 (Invitrogen, Clone: GK1.5, Catalog: 25-0041-82) and PE-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 12-0691-82) or PE-Cy5-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 15-0691-82), PE-anti-Foxp3 (Invitrogen, Clone: FJK-16s, Catalog: 12-5773-82), APC-anti-CD25 (Invitrogen, Clone: PC61.5, Catalog: 17-0251-82), PE-anti-CTLA-4 (Invitrogen, Clone: UC10-4B9, Catalog: 12-1522-82), PE-anti-ICOS (Invitrogen, Clone: C398.41, Catalog: 12-9949-81) or isotype controls(Invitrogen, USA) were used to analyze the phenotype of various Tregs. The expression levels of individual molecules in Tregs were determined using flow cytometry. Foxp3 staining was performed using the Foxp3 Fix/Perm buffer (Invitrogen, USA). After staining, cells were washed and fixed with 2% paraformaldehyde before analysis by flow cytometry. Post-analysis was performed using the FlowJo software.

Flow cytometry analysis was performed for lipid ROS measurement (Yu et al. 2023 (link)). HCC cells were grown in 6-well plates at 2 × 10⁵ cells/well. After treatment with 2 µM erastin or an equal volume of DMSO for 12 h, the culture medium received clearing and washing with PBS. Next, HCC cells received staining with BODIPY-C11 (5 µM) for 20 min at 37 °C. After washing twice with PBS and filtering with a 0.4-μM cell filter, flow cytometry was used to assess intracellular lipid ROS. The iTreg cells were generated in vitro. The transfected HCC cells received coculture with CD4⁺ T cells for 48 h, and proliferation of CD4⁺ T cells received assessment by CFSE staining using flow cytometry. Antibodies including APC-anti-CD4 or PE-Cy7-anti-CD4 APC-anti-CD25, PE-anti-FOXP3, PE-anti-CTLA4, PE-anti-TIGIT, PE-anti-TNFRSF4, or isotype controls provided by Invitrogen (USA) were utilized for analyzing the phenotype of various Tregs. FOXP3/CD25/CTLA4/TIGIT/TNFRSF4 staining was performed with Fix/Perm buffer (Invitrogen, USA). After staining, cells received washing and fixation with 2% paraformaldehyde before analysis through flow cytometry. The results were analyzed using FlowJo software. There are three biological replicates for flow cytometry analysis.

Mice were perfused for the analysis of brain sequestered cells. Brains were digested in RPMI containing collagenase (1.6 mg/mL; type IV; Sigma Aldrich) and DNaseI (200 μg/mL; Sigma Aldrich) at 37 °C for 50 min. Cells were isolated using a Percoll gradient (GE Healthcare). Debris was filtered out using a 70 μm nylon mesh. Cells were counted and labelled with LIVE/DEAD amine-reactive violet viability maker according to the manufacturer’s protocol (Invitrogen). The cells were blocked and labeled with FITC anti-CD45 (eBioscience; 30-F11), PE anti-CD11b (BD Pharmingen; M1/70), APC anti-CD4 (eBioscience; RM4-5) and PerCP-Cy5.5 anti-CD8 (eBioscience; 53–6.7). Flow cytometry was performed using a BD LSR Fortessa and results were analyzed using the FACS Diva 6.0 software.

Peripheral blood of mice was harvested by cardiac puncture technique after euthanasia into a tube containing heparin as anti-coagulant. Samples were diluted with saline in an equal volume and centrifuged at 400× g for 10 min at room temperature. After removal of upper plasma, the Ficoll^® Paque Plus buffer (17-1440-02, GE Healthcare Life Sciences, Shanghai, China) were mixed with the cell resuspension with saline in a 50-mL tube. After centrifugation at 800× g for 30 min at room temperature, the samples were separated into layers from top to bottom as platelet in plasma, peripheral blood mononuclear cells (PBMC), Ficoll buffer, and red blood cells. The upper layer of a sample was aspirated carefully and then collected PBMC composed of lymphocytes and monocytes predominantly. To count the frequencies of T lymphocytes by BD FACSCalibur™ platform (BD Biosciences, Taipei, Taiwan), cells were labeled with PE Hamster anti-CD3e (553063, BD Biosciences), APC anti-CD4 (17-0042, eBioscience, Thermo Fisher Scientific Inc., Waltham, MA), and FITC anti-CD8a (11-0081, eBioscience) to distinguish a subpopulation of T lymphocytes.

Flow cytometry was performed using a BD LSR Fortessa and results were analyzed using FlowJo version 9.6.2. Mice were perfused for the analysis of brain sequestered cells. Brains were digested in RPMI containing 1.6mg/mL collagenase (type IV; Sigma-Aldrich) and 200μg/mL DNase I (Sigma-Aldrich) at 37°C for 50 min. Cells were isolated using a Percoll gradient (GE Healthcare) and debris was filtered out using a 70μm nylon mesh. Cells were counted and labelled with LIVE/DEAD amine-reactive violet viability marker according to the manufacturer’s protocol (Invitrogen). The cells were blocked and labeled with FITC anti-CD45 (eBioscience; 30-F11), PE anti-CD11b (BD Pharmingen; M1/70), APC anti-CD4 (eBioscience; RM4-5) and PerCP-Cy5.5 anti-CD8 (eBioscience; 53–6.7).