Apc efluor780 conjugated anti cd8

In 24-well plates, naive CD4⁺ T cells were cultured for 15 or 30 min in the presence of anti-CD3 and anti-CD28 antibodies. For phospho-STAT5 staining, cells were washed extensively before the addition of 20 ng/ml recombinant mIL-2 (BD Biosciences) and incubated at 37°C for 10, 15, or 30 min. Stimulated cells were collected at indicated time points and stained with an APC-conjugated anti–phospho-STAT5 monoclonal antibody according to the manufacturer’s protocol C (Thermo Fisher Scientific). Additional surface staining was performed with APC-eFluor780–conjugated anti-CD8 (Thermo Fisher Scientific), PE-Cy7–conjugated anti-CD4 (Thermo Fisher Scientific), and live/dead cell viability staining. Intracellularly stained cells were analyzed using a MACSQuant Analyzer 10 (Miltenyi Biotec).

Single-cell suspensions prepared from CNS, iLNs, and spleens of immunized mice or from in vitro cultures were analyzed by ICS as previously described (Kim et al., 2017b (link)). Prepared cells were restimulated with 50 ng/ml PMA (Sigma-Aldrich) and 500 ng/ml ionomycin (Sigma-Aldrich) for 5 h at 37°C in a humidified 5% CO₂ atmosphere. After stimulation, the cells were stained with Fixable Viability Dye (Invitrogen) against cell surface proteins, APC-eFluor780–conjugated anti-CD8, PE-Cy7–conjugated anti-CD4, and AF700-conjugated anti-CD44 antibodies (Thermo Fisher Scientific). Following surface staining, samples were fixed and permeabilized using the Intracellular Fixation & Permeabilization buffer Set (Thermo Fisher Scientific). After fixation and permeabilization, the cells were stained with antibodies against FITC-labeled anti–IFN-γ (BD Bioscience), PE-labeled anti–IL-4 (BD Bioscience), and APC-labeled anti–IL-17A (Thermo Fisher Scientific). Appropriate fluorescein-conjugated and isotype-matched monoclonal antibodies were used as isotype controls. Flow cytometry was performed with either MACSQuant Analyzer 10 (Miltenyi Biotec) or LSR Fortessa (BD Biosciences), and data were analyzed with FlowJo software (BD Biosciences).