Anti cd3 apc

Intracellular cytokine measurement was performed according to Körholz et al., 2021 (7 (link)). Briefly PBMCs (1x10⁶/ml) were left unstimulated or stimulated with 10 ng/ml PMA (Phorbol-12-Myristat13-Acetat) and 1µg/ml Calcium-Ionophore (both Merck KGaA, Darmstadt, Germany) under addition Brefeldin A (1µl/ml, BD-Biosciences San José, CA) for 12h overnight. Cells then were harvested and washed twice with PBS/1%FCS. For surface staining, cells were incubated with anti-CD45RO-PE (5µl/test) (Biolegend, San Diego, CA), anti-CD3-APC (2µl/test), anti-CD4-APC-AlexaFluor750 (5µl/test) and anti-CD45-Krome Orange (5µl/test) (Beckman Coulter, Krefeld, Germany) for 30 min at 4°C. After beeing washed twice with PBS/1% FCS, cells were fixed and permeabilized with 100µl Cytofix/Cytoperm™ (BD Biosciences, San José, CA) for 20 min at 4°C and then washed twice according to the manufacturers instructions. For intracellular staining, cells were incubated with anti-IFNγ-FITC (5µl/test), anti-IL4-PE Cy7 (5µl/test) (both Biolegend) and anti-IL-17A eFlour 450 (5µl/test) (eBioscience/Thermo Scientific) and anti-CD4 APC-AlexaFluor750 (2µl/test) (Beckman-Coulter, Krefeld,Germany) for 45min at 4°C. Cells were washed twice with Perm/Wash Buffer (BD Biosciences) and diluted in 500µl PBS/1% FCS. Analysis was performed on a BD LSR II.