Fitc anti cd4 antibody

Leukocyte activation cocktail (2 μl/ml) stimulated the CD4+ T cells (1 × 106 cells/ml), cultured for 6 hours in 5% CO2 at 37°C, and finally collected for cytometry analysis. Then, cells were stained with cell viability marker for 15 mins at room temperature in dark after 6 hours of incubation. The cells were stained with FITC-anti-CD4 antibody (Biolegend) for surface markers, fixation, and permeabilization performed by Cytofix/Cytoperm Soln Kit (BD Pharmingen) in dark at 4°C for half an hour, and then washed with permeabilization buffer and stained with PE-anti-IL-4 and APC-anti-IL-17A antibodies in permeabilization buffer at 4°C in the dark for half an hour. Finally, flow cytometry was performed, and data were analyzed using FACS Canto II (Becton Dickinson) and FlowJo version X software.

CD4⁺ T cells were stimulated with 2 μL/ml (1 × 10⁶ cells/ml) leukocyte activation cocktail (550583, BD Biosciences, United States ), and cultured at 37°C with 5% CO₂ for 6 h, then collected for flow cytometry analysis. After a 6-h incubation, cells were stained with a marker of cell viability (Fixable Viability Stain 510 antibody, BD Pharmingen) for 15 min at room temperature in the dark. Then, cells were stained for surface markers with FITC-anti-CD4 antibody (Biolegend) followed by fixation and permeabilization using the Cytofix/Cytoperm Soln Kit (BD Pharmingen) for 30 min at 4°C in the dark. After washing with permeabilization buffer, cells were stained for intracellular markers with APC-anti-IL-17 A (Biolegend) and PE-anti-IL-4 (BD Pharmingen) antibodies in permeabilization buffer for 30 min at 4°C in the dark. Isotype controls were employed in the control group. Flow cytometry was performed, and data were analyzed using FACS CantoⅡ(Becton Dickinson) and FlowJo version X software.

Flow cytometry was performed to assess T cell phenotypes using the following antibodies: FITC anti-CD4 antibody (357406, BioLegend, San Diego, CA, USA), APC-Cy7 anti-CD8 antibody (344714, BioLegend), PE anti-CD45RO antibody (304206, BioLegend), and APC anti-CCR7 (353214, BioLegend). Stained cells were tested by flow cytometry in a staining buffer (phosphate-buffered saline (PBS) with 2% fetal bovine serum). Data were analyzed using Flowjo V10 software.