Flow Cytometry Analysis of Immune Cells

Flow cytometry was performed as described (10 (link), 15 (link)). Cells were incubated with anti-mouse CD16/32 (Fc Block; BD Biosciences) before staining with primary antibody or isotype control antibodies (10 min, 22°C). Ten-thousand to 50,000 events per sample were acquired using a CYAN ADP flow cytometer (Beckman-Coulter) and analyzed with FCS Express 3 (De Novo Software). The following antibodies were used: anti-ScaI-phycoerythrin (PE), anti-TNFα-allophycocyanin, anti-Ly6G-PE, anti-B220-allophycocyanin-CY7, anti-CD11c-allophycocyanin, anti-B220-FITC, anti-Ly6C-FITC, anti-CCR7-PE (BD Bioscience), avidin-PE-CY7, anti-CD11b-Brilliant Violet, anti-CCR9-FITC, and anti-PDCA-1-biotin (BioLegend). Intracellular TNFα staining was performed as described (16 (link)).

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Xu Y., Zhuang H., Han S., Liu C., Wang H., Mathews C.E., Massini J., Yang L.J, & Reeves W.H. (2015). Mechanisms of TNFα antagonist-induced lupus in a murine model. Arthritis & rheumatology (Hoboken, N.J.), 67(1), 225-237.

Publication 2014

anti-mouse CD16/32 (Fc Block; CYAN ADP flow cytometer FCS Express 3 anti-Ly6G-PE anti-B220-FITC anti-Ly6C-FITC anti-CCR7-PE

Allophycocyanin Antibodies Antibody Avidin Biotin Ccr9 Cd11b Cells Fitc Flow cytometry Intracellular Isotype Mouse Pdca 1 Phycoerythrin Tnfα Violet

Corresponding Organization :

Other organizations : University of Florida

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Variable analysis

independent variables

Anti-mouse CD16/32 (Fc Block; BD Biosciences)
Primary antibody or isotype control antibodies

dependent variables

Percentage of cells expressing the following markers: Sca1, TNFα, Ly6G, B220, CD11c, Ly6C, CCR7, CD11b, CCR9, PDCA-1

control variables

Cell incubation time (10 min)
Cell incubation temperature (22°C)
Acquisition of 10,000 to 50,000 events per sample using a CYAN ADP flow cytometer (Beckman-Coulter)
Analysis using FCS Express 3 (De Novo Software)

controls

Isotype control antibodies

Annotations

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