Blood eosinophils were activated with IL-3 (2ng/ml), IL-5 (4ng/ml) or GM-CSF (2ng/ml), or cultured without cytokine (Resting) for 20 h and then stained for flow cytometric analysis. PE-conjugated anti-CD32 (FCGRII) (clone FUN-2) was from Biolegend (San Diego, CA), FITC-conjugated anti-CD64 (FCGRI-clone 10.1), FITC-conjugated anti-CD16 (FCGRIII-clone 3G8), PE-conjugated anti CD11b (αM-clone D12), FITC-conjugated anti-CD18 (ß2-clone L130) and corresponding isotype controls, PE- and FITC-conjugated mouse IgG1 were all from BD Biosciences. The activation state of CD32 was measured using the previously described monoclonal phage antibody A17 [34 (link), 35 (link)]. Activation-sensitive anti-αM CBRM1/5 was from BioLegend and anti-β1 N29, from Chemicon/Millipore/Sigma-Aldrich [36 (link)]. PE-conjugated goat anti-mouse Ab was used as the secondary Ab. Five to 10 thousand viable (no propidium iodide uptake) cells were acquired on a FACSCalibur (BD Biosciences). Data were analyzed with FlowJo (TreeStar Inc., Ashland, OR).
Protocol detail
Cytokine-induced Eosinophil Activation
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Blood eosinophils
Cd11b
Cells
Clone
Cytokine
Fitc
Flow cytometric
Gm csf
Goat
Igg1
Isotype
Monoclonal antibody
Mouse
Phage
Propidium iodide
Corresponding Organization : University of Wisconsin–Madison
Other organizations : University Medical Center Utrecht
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Variable analysis
independent variables
- IL-3 (2ng/ml)
- IL-5 (4ng/ml)
- GM-CSF (2ng/ml)
- Resting (no cytokine)
- Activation state of CD32 measured using monoclonal phage antibody A17
- Expression of CD32 (FCGRII), CD64 (FCGRI), CD16 (FCGRIII), CD11b (αM), CD18 (β2)
- Incubation time (20 h)
- Flow cytometric analysis
- Activation-sensitive anti-αM CBRM1/5
- Anti-β1 N29
- Corresponding isotype controls for antibodies (PE- and FITC-conjugated mouse IgG1)
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