Pe cy5 coupled anti cd3

For determination of phosphorylated proteins after CD40 stimulation, PBMCs were thawed and rested for 2 h and then either left unstimulated or stimulated with trimeric CD40L (100 ng/ml) (Enzo Life Sciences, Farmingdale, NY) for 15 min, performed concurrently with decoration with V450-coupled anti-CD19 (Catalog#: 560353, BD Biosciences). After incubation with BD Cytofix^™ Fixation Buffer (Catalog#: 554655) for 10 minutes at 37°C;, followed by permeabilization in BD Phosflow^™ Perm Buffer III (Catalog#: 558050) for 30 minutes on ice, cells were then stained with PE-Cy5-coupled anti-CD3 (BioLegend), FITC-coupled anti-pp38 (Catalog#: 4551, Cell Signaling Technology, Danvers, MA), PE-coupled anti-pp65 (Catalog#: 558423, BD Biosciences), or PE-coupled anti-pErk (Catalog#: 612566, BD Biosciences). The analysis was performed using an LSR II flow cytometer (BD Biosciences) and FlowJo 7.6.2 software (TreeStar, Ashland, OR).

PBMCs were thawed, washed by centrifugation, and 5 × 10⁵ cells were aliquotted to each tube. Dead cells were excluded from the analysis by staining with LIVE/DEAD Fixable Near-IR Dead Cell Stain (Catalog#: L10119, Life Technologies, Waltham, MA). Antibodies to cell-surface markers were mixed and added to the cells, including PE-Cy5-coupled anti-CD3 (5 μl per 10⁶ cells; Catalog#: 344808, BioLegend, San Diego, CA), PE-coupled anti-CD19 (Catalog#: 12-0199-41, eBioscience, Santa Clara, CA), V450-coupled anti-CD14 (Catalog#: 560349, BD Biosciences, San Jose, CA), PE-Cy7-coupled anti-CD4 (5 μl per 10⁶ cells; Catalog#: 300512, BioLegend) and V500-coupled anti-CD8 (Catalog#: 560774, BD Biosciences). After incubation for 30 min at 4°C;, the cells were washed twice with BD Pharmingen^™ Stain Buffer (Catalog#: 554656). The cells were then fixed and permeabilized for 60 min at 4°C; using the Fixation/Permeabilization diluent and concentrate (Catalog#: 00-5123-43 and 00-5223-56, eBioscience). Cells were then stained with eFluor 660-coupled anti-Act1 (Catalog#: 50-4040, eBioscience) or eFluor 660-coupled isotype control (eBioscience). The analysis was performed using flow cytometer (BD LSR II, BD Biosciences) and FlowJo 7.6.2 (TreeStar, Ashland, OR) software.