3168008b 169tm cd25 2a3 fluidigm 3169003b 170er cd45ra hi100 fluidigm 3170010b 171yb cd195 np 6g4 fluidigm 3171017a  (fluidigm)

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: 170 Er , CD3 , Standard BioTools , 3170001B , UCHT1 , 17.5.

Techniques: Staining, Expressing

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: This bar graph illustrates the average frequency of total (%) of the major immune cell subsets in donor-matched ( A ) cPBMCs and ( B ) whole blood from (n = 3) healthy individuals. The data points represent the average frequency of total (%) of each subset, with error bars indicating the standard deviation from triplicate samples. Major immune subsets, including B cells, monocytes, dendritic cells (DCs), NK cells, NKT cells, and T cells, were identified using a comprehensive high-dimensional immunophenotyping strategy defined after Gaussian parameter cleanup (Figure 2) and doublet exclusion. These lineages were identified through expression of markers such as CD3, CD19, CD56, CD14, CD11c, HLA-DR, and CD66b, following the detailed gating strategy outlined in Figure 3. As expected, cPBMCs have a higher proportional representation of lymphocytes (T cells, B cells, NK, and NKT cells) and monocytes but lack granulocytes, whereas whole blood maintains the complete cellular repertoire, including a larger proportion of granulocytes and what appears to be a lower percentage of lymphocyte or monocyte subsets due to being expressed as a percentage of total leukocytes.

Article Snippet: 170 Er , CD3 , Standard BioTools , 3170001B , UCHT1 , 17.5.

Techniques: Standard Deviation, Expressing