Mixed cell suspensions were stained for surface markers with combinations of the following antibodies: CD45-vioblue450, CD11b-PE (Tonbo, San Diego, CA), CD3-VioGreen, CD15-FITC, CD66b-APC (Miltenyi biotec), CD11c-PerCp-Cy5.5, CD1c-PE-dazzle, HLA-DR-BV570, CD3-APC (Biolegend), CD14-e780, (eBiosciences, San Diego, CA). Dead cells were excluded with 7AAD (Southern Biotech) or zombie dye yellow staining (Biolegend). Analysis was performed on 8-color MACSQuant 10 (Miltenyi biotech) or Gallios (Beckman Coulter, Indianapolis, IN) flow cytometers and data analyzed with FlowJo software (Tree Star, Inc. Ashland, OR). Expression of surface markers is shown as percentage of positive cells. Fluorescence minus one (FMO) strategy was used to establish appropriate gates. Cells were gated as described before26 (link).
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Multi-Parametric Flow Cytometry of Immune Cells
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Corresponding Organization : Dartmouth College
Other organizations : University of Alabama at Birmingham
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Variable analysis
independent variables
- CD45-vioblue450
- CD11b-PE
- CD3-VioGreen
- CD15-FITC
- CD66b-APC
- CD11c-PerCp-Cy5.5
- CD1c-PE-dazzle
- HLA-DR-BV570
- CD3-APC
- CD14-e780
- Expression of surface markers is shown as percentage of positive cells
- 7AAD (Southern Biotech) or zombie dye yellow staining (Biolegend) used to exclude dead cells
- Fluorescence minus one (FMO) strategy used to establish appropriate gates
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