CD23 Monoclonal Antibody (EBVCS2), eBioscience™

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Aliquots of 1.5 × 106 cells were used for each staining.	Before staining, cells were blocked with 10% vol/vol mouse serum (Life Technologies, Carlsbad, Calif).	Dead cells were excluded from the analysis with eFluor 780 Fixable Viability Dye (eBioscience). Flow cytometric analysis was performed on a Beckman Coulter FC 500 flow cytometer (Beckman Coulter).

Upstream tips
Aliquots of 1.5 × 106 cells were used for each staining.
Protocol tips
Before staining, cells were blocked with 10% vol/vol mouse serum (Life Technologies, Carlsbad, Calif).
Downstream tips
Dead cells were excluded from the analysis with eFluor 780 Fixable Viability Dye (eBioscience). Flow cytometric analysis was performed on a Beckman Coulter FC 500 flow cytometer (Beckman Coulter).

Publication protocol

Red blood cell lysis solution (155 mmol/L ammonium chloride, 10 mmol/L potassium bicarbonate, and 12 mmol/L EDTA) was applied to heparinized blood samples from patients. For flow cytometry, the following surface markers of cells were stained: T cells (positive with anti-CD3, clone OKT3), natural killer (NK) cells (negative with anti-CD3 and positive with anti-CD56, clone TULY56), B cells (positive with anti-CD19, clone HIB19), monocytes (positive with anti-CD14, clone 61D3), platelets (positive with anti-CD61, clone VI-PL2, and with anti-CD41a, clone HIP8), basophils (positive with anti-CD123, clone 6H6, and anti-CCR3, clone 5E8-G9-B4), neutrophils (granulocytes negative with anti-CD49d, clone HP2/1), eosinophils (granulocytes positive with anti-CD49d, clone HP2/1, and negative with anti-CD19), dendritic cells (lineage cocktail negative, positive with anti-CD11c, clone 3.9), naive B cells (positive with anti-CD19, clone J3-119, and anti-IgD, clone 11-26), memory B cells (positive with anti-CD19, clone J3-119, and positive with anti-CD27, clone O323). All cells were additionally stained with anti-CD23 (clone EBVCS2). Matching nonbinding isotype antibodies were used as controls. All antibodies were obtained from eBioscience (San Diego, Calif), except for anti-CD49d, anti-CD19 for naïve/memory staining (Beckman Coulter, Brea, Calif), and lineage cocktail lin1 (BD Biosciences, Franklin Lakes, NJ). Aliquots of 1.5 × 106 cells were used for each staining. Before staining, cells were blocked with 10% vol/vol mouse serum (Life Technologies, Carlsbad, Calif). Dead cells were excluded from the analysis with eFluor 780 Fixable Viability Dye (eBioscience). Flow cytometric analysis was performed on a Beckman Coulter FC 500 flow cytometer (Beckman Coulter). Depending on the cell type, 3 × 105 (T cells, B cells, NK cells, and monocytes), 1 × 106 (basophils), or 5 × 105 (all other cell types; ie, neutrophils, eosinophils, dendritic cells, and platelets) events were recorded. FlowJo Software 7.5 (TreeStar, Ashland, Ore) was used for data analysis. Gates were set according to the matching nonbinding isotype control of each antibody for each cell type.

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