Journal: Bio-protocol
Article Title: Dual Phospho-CyTOF Workflows for Comparative JAK/STAT Signaling Analysis in Human Cryopreserved PBMCs and Whole Blood
doi: 10.21769/BioProtoc.5512
Figure Lengend Snippet: The sequential gating strategy was used to identify immune cell populations from healthy human samples. Healthy donor samples, either ( A ) cryopreserved peripheral blood mononuclear cells (cPBMCs) or ( B ) fresh whole blood (WB), were stimulated for 15 min, fixed, palladium-barcoded, and stained with a comprehensive panel of 19 (for cPBMCs) or 20 (for whole blood) surface markers. Within the single-cell gate, non-granulocytes (leukocytes) were defined as CD66b-CD45 + , while granulocytes were confirmed solely in whole blood as CD66b + CD45-. Lymphocytes were resolved using CD3 and CD56 [T cells (CD3 + CD56-)], NKT cells (CD3 + CD56 + ), and NK cells (CD3-CD56 + ). B cells were identified as CD19 + CD20 +/- within the CD3-CD56- gate, further confirmed by negative expression of CD123 and CD11c. Both T and B cells were further subset based on the expression of CD4, CD8, CD45RA, CD27, and CD25 or IgD and CD27, respectively. Non-T, non-B, and non-NK cells (CD45 + CD3-CD19-CD56-CD20-) were separated based on their expression of CD11c and HLA-DR. Total DCs were defined as CD11c + HLA-DR + and can be further defined through the expression of CD123 (plasmacytoid DCs). Monocytes were resolved within this non-lymphocyte gate based on their expression of CD14 and CD16.
Article Snippet: 145 Nd , CD4 , Standard BioTools , 3145001B , RPA-T4 , 35.
Techniques: Staining, Expressing