Comprehensive Immune Cell Profiling by Flow Cytometry

Flow cytometry was performed as previously described [38 (link)]. Briefly, cells were incubated with LIVE/DEAD Fixable cell stain (Invitrogen), then stained for 20 min with: MHCII-PacificBlue, F4/80-APC (Biolegend), CD4-AF700, CD8-PacificBlue, CD25-APC, CD86-APC, CD49b-Pe-Cy7 (eBioscience), CD11b-PE-Cy7, CD45R/B220-PerCP-Cy5.5 (BD-Pharmingen). For intracellular staining, the samples were stimulated for 3 h (100 ng/ml phorbol 12-myristate 13-acetate, 1 μg/ml ionomycin; monensin added after 1 h). Cells were fixed and permeabilized with Cytofix/Cytoperm (BD), then stained with IL-10-FITC, IFN-γ-PerCP-Cy5.5, IL-17-PE, FoxP3-AF488 (BD-Pharmingen) antibodies before analysis on LSRII flow cytometer (BD), collecting 100,000 live events. Data was analyzed using FlowJo (v7.6.5, Tree Star, Ashland, OR, USA).

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Schuijs M.J., Hartmann S., Selkirk M.E., Roberts L.B., Openshaw P.J, & Schnoeller C. (2016). The Helminth-Derived Immunomodulator AvCystatin Reduces Virus Enhanced Inflammation by Induction of Regulatory IL-10+ T Cells. PLoS ONE, 11(8), e0161885.

Publication 2016

MHCII-PacificBlue F4/80-APC CD4-AF700 CD8-PacificBlue CD25-APC CD86-APC CD49b-Pe-Cy7 CD11b-PE-Cy7 CD45R/B220-PerCP-Cy5.5 Cytofix/Cytoperm IFN-γ-PerCP-Cy5.5 IL-17-PE FoxP3-AF488 LSRII flow cytometer

Acetate Antibodies Cd11b Cd25 Cells Cy5 5 Fitc Flow cytometry Ifn γ Il 10 Il 17 Intracellular Ionomycin Monensin Phorbol 12 myristate Tree

Corresponding Organization : Freie Universität Berlin

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Variable analysis

independent variables

Treatment with phorbol 12-myristate 13-acetate (100 ng/ml)
Treatment with ionomycin (1 μg/ml)

dependent variables

Expression of IL-10, IFN-γ, IL-17, FoxP3 (measured by intracellular staining)
Percentage of live cells expressing MHCII, F4/80, CD4, CD8, CD25, CD86, CD49b, CD11b, CD45R/B220 (measured by flow cytometry)

control variables

Incubation time for stimulation (3 h)
Monensin addition after 1 h of stimulation
Cell fixation and permeabilization protocol
Flow cytometry instrument (LSRII)

controls

Positive control: Cells stimulated with phorbol 12-myristate 13-acetate and ionomycin
Negative control: Unstimulated cells

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