Alexa700 conjugated anti cd3

Cryopreserved PBMC were thawed in warm media (RPMI 1640 supplemented with 10 % fetal bovine serum, 1 % penicillin-streptomycin, 10 mM HEPES, 2 mM L-glutamine (all Hyclone), and 10 μg/ml DNAse I (Sigma)), washed, and stained for viability with a Yellow Amine Reactive Dye (YARD) for 15 min at room temperature. To identify monocytes, the cells were then surface-stained for 30 min with V500-conjugated anti-CD3, Qdot605-conjugated anti-CD14, Alexa700-conjugated anti-CD16, PE-Cy7-conjugated anti-CD56, PE-Cy7-conjugated anti-CD19, PE-Cy7-conjugated anti-CD20, and APC-H7-conjugated HLA-DR monoclonal antibodies (mAbs). All antibodies were from BD Biosciences except for Q605-conjugated anti-CD14 and yellow Live/Dead (Life Technologies). Alexa700-conjugated anti-CD3, APCH7-conjugated anti-CD8, V450-conjugated anti-CD38, and APC-conjugated anti-HLA-DR all mAbs (all from BD Biosciences) were used to define T cell activation. Fluorescence minus one samples were prepared for each fluorochrome to facilitate gating. All cells were fixed with 1 % PFA in PBS, and analyzed by flow cytometry using a four-laser custom BD-Fortessa instrument (Becton Dickinson). A total of 100,000 cells was collected and analyzed with FlowJo software (TreeStar).