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Ten-parameter immunophenotyping of human PBMCs with the Attune® NxT Acoustic Focusing Cytometer.
Workflow: Red blood cells were lysed from human whole blood and PBMCs were surface stained with the following anti-human antibodies: CD45 Pacific Orange™ (Cat. no. MHCD4530 ), CD4 PerCP-Cy®5.5 (Cat. no. A15858), CD20 APC, CD14 APC-Cy®7 (Cat. no. A15453), CD19 Pacific Green™ (Cat. no. C11210), CD3 Alexa Fluor® 700 (Cat. no. CD0329), HLA-DR PE-Cy®7 (Cat. no. A18558), CD8 Pacific Blue™ (Cat. no. MHCD0828), CD33 FITC (Cat. no. A16185), and CD11c PE (Cat. no. A18674). Data Acquisition: Samples were collected using the Attune® NxT Acoustic Focusing Cytometer with 405 nm excitation and 440/50 bandpass emission filter (Pacific Blue™), 512/25 bandpass emission filter (Pacific Green™), and 603/48 bandpass emission filter (Pacific Orange™); 488 nm excitation and 530/30 bandpass emission filter (FITC) and 695/50 bandpass emission filter (PerCP-Cy®5.5); 561 nm excitation and 585/16 bandpass emission filter (PE) and 780/60 bandpass emission filter (PE-Cy®7); 637 nm excitation and 660/20 bandpass emission filter (APC), 720/30 bandpass emission filter (Alexa Fluor®700), and 780/60 bandpass emission filter (APC-Cy®7). Gating Strategy: Initial gates were drawn on monocytes and lymphocytes based on forward and side scatter profiles. Within the lymphocyte gate, T cells can be isolated based on their expression of CD3 and further separated into CD4 and CD8 subpopulations. Within the CD3 negative CD45 positive gate, B cells can be identified based on expression of CD19 and CD20. Conventional dendritic cells found in peripheral blood are generally negative for T and B cell lineage markers and co-express the integrin CD11c and HLA-DR. Values noted are a percent of the parent CD19 negative CD20 negative gate (top value, no parenthesis) or percent of FSC/SSC lymphocyte gate (bottom value, in parenthesis).
