PBMCs were stained in 96-well U-bottomed plates as described previously [6 (link)]. Briefly, cells were stained with fluorophore-labelled antibodies to cell surface markers, fixed, permeabilised (Cytofix/Cytoperm; BD Biosciences), and stained for intracellular molecules. The following monoclonal antibodies were used: anti-CD3-V500, anti-CD56-PECy7, anti-IFN-γ-APC, anti-CD107a-FITC, anti-CD16-APC-H7, anti-CD25-APC-H7, (all BD Biosciences), anti-CD57-e450, anti-CD25-PErCPCy5.5, anti-CD16-APC, anti-CD25-PE, anti-IL18Rα-PE, anti-IL18Rα-FITC, anti-IFN-γ-APCe780, anti-CD16-APCe780 (all e-Biosciences), anti-NKG2C-APC, anti-NKG2C-PE (both R&D Systems), and anti-NKG2A-FITC (Miltenyi). IL-12Rβ2 antibody was conjugated using EasyLink PE-Cy5 (AbCam). Cells were acquired on an LSRII flow cytometer (BD Biosciences) using FACSDiva® software. Data analysis was performed using FlowJo V10 (Tree Star). FACS gates set on unstimulated cells (medium alone or isotype controls) were applied in standard format across all samples and all conditions.
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Comprehensive PBMC Immunophenotyping Protocol
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Other organizations : University of London, London School of Hygiene & Tropical Medicine
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Variable analysis
independent variables
- Fluorophore-labelled antibodies to cell surface markers
- Expression of cell surface markers
- Expression of intracellular molecules
- PBMCs stained in 96-well U-bottomed plates
- Cells fixed and permeabilised
- Unstimulated cells (medium alone or isotype controls) used to set FACS gates
- Unstimulated cells (medium alone or isotype controls) used as negative controls
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