Comprehensive Immune Cell Profiling Protocol

PBMCs were thawed, washed and incubated with Gamunex and EMA, then stained with a cocktail of lineage (Lin) markers (CD3, CD19, CD56)-Brilliant Violet 605 (BioLegend, BD-Biosciences), CD14-Brilliant Violet711, CD11c-PE-Cy7 (BioLegend), CD45-V500, CD123-BV421, HLA-DR-PerCP-Cy5.5, CD15-FITC, CD11b-APC-Cy7, CD33-Alexa Fluor 700 (BD-Biosciences), and CD124-APC (R&D Systems) using EMA to identify dead cells.
To characterize Tregs, we used the same panel as before [23 (link)], staining PBMCs forCD3 (OKT3 supernatant) with Pacific Orange-conjugated secondary antibody (Invitrogen) followed by staining for CD4-Pacific Blue, CD45RA-Alexa Fluor-700, CD8-Peridinin-chlorophyll protein (PerCP), CD279-PerCP-Cy5.5, CD127-Alexa Fluor-647 (Bio legend), CD25-APC-Cy7 (BD Biosciences) and intracellular staining for FoxP3-PE (Bio legend). All samples were measured using a BD LSRII (BD Biosciences) immediately after staining.

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Kini Bailur J., Gueckel B, & Pawelec G. (2016). Prognostic impact of high levels of circulating plasmacytoid dendritic cells in breast cancer. Journal of Translational Medicine, 14, 151.

Publication 2016

CD56)-Brilliant Violet 605 CD14-Brilliant Violet711 CD11c-PE-Cy7 CD45-V500 CD123-BV421 HLA-DR-PerCP-Cy5.5 CD15-FITC CD11b-APC-Cy7 CD33-Alexa Fluor 700 CD124-APC Pacific Orange-conjugated secondary antibody CD45RA-Alexa Fluor-700 CD127-Alexa Fluor-647 CD25-APC-Cy7 BD LSRII

Alexa fluor 647 Antibody Cd11b Cd123 Cd124 Cd25 Cd279 Cells Chlorophyll Cy5 5 Fitc Gamunex Hla dr Intracellular Okt3 Peridinin Protein Violet

Corresponding Organization :

Other organizations : Nottingham Trent University

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Variable analysis

independent variables

Gamunex

dependent variables

Percentage of cells expressing CD3, CD19, CD56, CD14, CD11c, CD45, CD123, HLA-DR, CD15, CD11b, CD33, CD124
Percentage of cells expressing CD3, CD4, CD45RA, CD8, CD279, CD127, CD25, FoxP3

control variables

PBMCs
Staining with a cocktail of lineage markers
Measurement using a BD LSRII flow cytometer

positive controls

Not specified

negative controls

EMA to identify dead cells

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