Computational protocol: IL-33-Induced Cytokine Secretion and Survival of Mouse Eosinophils Is Promoted by Autocrine GM-CSF

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Protocol publication

[…] BMDE or BMDM were harvested and washed once in FACS buffer (PBS/2% FCS/1 mg/ml sodium azide), incubated with anti-CD16/CD32 blocking mAb (2.4G2, BioXcell) for 5 min at room temperature, and stained with diluted mAb mixtures. The following mAbs were used: PE- or Alexa647-coupled anti-mouse-Siglec-F (clone E50-2440, BD Biosciences, San Jose, CA) to determine BMDE purity and Alexa488-coupled anti-mouse-CD11b and APC/Cy7-coupled anti-mouse-F4/80 to determine BMDM purity. Apoptotic cells were stained with APC-Annexin V (Immunotools) in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2) according to manufacturer’s protocol. Propidium iodide (eBioscience, San Diego, CA) was added to identify dead cells. Samples were acquired on a FACS Canto II instrument (BD Immunocytometry Systems, San Jose, CA) and analyzed by FlowJo software (Tree Star, Ashland, OR). [...] Student′s t-test was performed with SigmaPlot (Systat Software Inc., San Jose, CA, USA) software and P-values of less than 0.05 were considered statistically significant. […]

Pipeline specifications

Software tools FlowJo, SigmaPlot
Applications Miscellaneous, Flow cytometry
Organisms Mus musculus
Diseases Drug Hypersensitivity