Horizon v450 conjugated anti cd4

Bone marrow cells were harvested by flushing 5 ml PBS through the bone cavity of one femur using a syringe. After centrifugation at 473 g for 5 min, cells were resuspended in Tris-buffered 0.83% NH₄Cl solution (pH 7.29) for 5 min to lyse erythrocytes and then washed in PBS. For flow cytometry analyses, cells were extracellularly stained with BD Horizon v450-conjugated anti-CD4 (Becton Dickinson (BD), Franklin Lakes, NJ, USA) and allophycocyanin (APC) anti-CD25 (BD). By using anti-Mouse Foxp3 Staining Set (eBioscience, Vienna, Austria), Foxp3 was intracellularly stained with Phycoerythrin (PE)-conjugated anti-Foxp3, according to the manufacturer's instructions. Regulatory T cells were defined as CD4⁺CD25⁺Foxp3⁺ and results are expressed as frequency of lymphocyte parent gate. Samples were run on a BD FACS Canto II and data was further processed using Flow Jo 8.8.6 software (Three Star Inc, Ashland, USA).

Isolated splenocytes were stained for analysis by using the following antibodies: BD Horizon V450-conjugated anti-CD4 (Becton Dickinson) and fluorescein isothiocyanate (FITC)-conjugated anti-CD8 (Becton Dickinson). Synovial cells were examined by using BD Horizon V450-conjugated anti-CD11b (Becton Dickinson), FITC-conjugated anti-CD3 (Becton Dickinson), allophycocyanin (APC)-conjugated anti-F4/80 (BioLegend, San Diego, CA, USA), Peridinin Chlorophyll Protein Complex (PerCP)-conjugated anti-CD19 (BioLegend), and phycoerythrin (PE)-conjugated anti-Gr1. Labeled cells were analyzed in a FACS CantoII (Becton Dickinson). FlowJo version 8.5.2 (Tree Star, Ashland, OR, USA) software was used for analyzing the data.