3145001b  (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: 145 Nd , CD4 , Standard BioTools , 3145001B , RPA-T4 , 35.

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: The distinct phospho-STAT responses across major immune cell subsets found in (n = 3) healthy donor cPBMCs with two technical replicates per donor after 15 min of stimulation with IFNα (1e4 U/mL), IFNγ (50 ng/mL), and IL-21 (50 ng/mL). ( A ) Heatmaps display the fold change in phosphorylation for STAT1, STAT3, STAT4, STAT5, and STAT6 across major immune subsets. Fold change for each intracellular cell-state marker was calculated using the formula MMI stim /MMI unstim for each donor and displayed as the average of duplicates. ( B ) Bar graphs illustrating the average fold change over unstimulated (mean ± SD) in phosphorylation for each STAT protein within key immune cell populations [B cells, Monocytes, dCs, NK cells, NKT cells, and T cells (CD4 + and CD8 + )] across three donors.

Article Snippet: 145 Nd , CD4 , Standard BioTools , 3145001B , RPA-T4 , 35.

Techniques: Phospho-proteomics, Marker

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 distinct phospho-STAT responses across major immune cell subsets found in donor-matched (n = 3) whole blood with two technical replicates per donor after 15 min of stimulation with IFNα (1e4 U/mL), IFNγ (50 ng/mL), and IL-21 (50 ng/mL). ( A ) Heatmaps display the fold change in phosphorylation for STAT1, STAT3, STAT4, STAT5, and STAT6 across major immune subsets. Fold change for each intracellular cell-state marker was calculated using the formula MMI stim /MMI unstim for each donor and displayed as the average. ( B ) Bar graphs illustrating the average fold change over unstimulated (mean ± standard deviation) in phosphorylation for each STAT protein within key immune cell populations [granulocytes, monocytes, DCs, B cells, NK cells, NKT cells, and T cells (CD4 + and CD8 + )] across all three donors.

Article Snippet: 145 Nd , CD4 , Standard BioTools , 3145001B , RPA-T4 , 35.

Techniques: Phospho-proteomics, Marker, Standard Deviation