Cd25 allophycocyanin apc

Primary CD4⁺ T cells isolated from buffy coats of healthy volunteers were treated with different concentration of 5-iodotubercidin, topiramate, and trametinib and PMA/ionomycin as a positive control. After 24 and 48 h, cells were stained with annexin V to examine the percentage of cells undergoing apoptosis and with the surface receptor CD69 and CD25 to measure T cell activation. For staining, 10⁶ cells were washed with PBS supplemented with 3% FBS and 2.5 mM CaCl₂ followed by staining with annexin V-phycoerythrin (PE) (Becton Dickinson), CD69-fluorescein isothiocyanate (FITC) (eBioscience), and CD25-allophycocyanin (APC) (eBioscience) for 20 min at 4°C in the presence of 2.5 mM CaCl₂. Cells were then washed with PBS-FBS-CaCl₂ and analyzed by flow cytometry. Between 2 × 10⁵ and 4 × 10⁵ events were collected per sample within 3 h after staining on an LSRFortessa (BD Biosciences, 4 lasers, 18 parameters) and analyzed using FlowJo software (version 9.7.4, TreeStar).

Cell pellets were prepared from the spleen tissues isolated from PIAS3- and Mock-treated mice. Populations of Th cells were examined by staining the tissues with a monoclonal Ab against CD4–peridin chlorophyll protein (perCP) and CD25-Allophycocyanin (APC) (eBioscience). Cells were permeabilized and fixed with CytoFix (BD Biosciences, San Jose, CA, USA) as instructed by the manufacturer, and stained with Abs against IL-17–FITC, IFNγ-PE, Foxp3-FITC (eBioscience), and PIAS3 APC (abcam, MA, USA). Flow cytometric analysis was primarily gated of CD4, then percentage of IL-17 positive, interferon-γ positive, Foxp3/CD25, and PIAS3 positive cells were calculated.