Eosinophils were generated from the bone marrow of WT, Siglec8+F+, Siglec8+F− and SiglecF−/− mice following a protocol previously described by Dyer et al.37 (link) The surface expression of Siglec-F, Siglec-8 and CCR3 on bone marrow cells throughout the differentiation process was measured with flow cytometry as done previously.14 (link) Cell viability was assessed either with DAPI (ThermoFisher Scientific) or Ghost fixable viability dye (Tonbo Biosciences, San Diego, CA). Mature eosinophils on day 14 of the differentiation protocol were used for functional assays or apoptosis testing. Cytospins of mature eosinophils were stained with a Diff- Kwik™ stain set (Shandon, ThermoFisher Scientific, Waltham, MA) and imaged on an Olympus DSU microscope (Olympus, Tokyo, Japan).
Protocol detail
Eosinophil Differentiation from Murine Bone Marrow
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Apoptosis
Assays
Bone marrow
Bone marrow cells
Ccr3
Cell viability
Dapi
Eosinophils
Flow cytometry
Ghost
Mice
Microscope
Siglec
Stain
Corresponding Organization : Northwestern University
Other organizations : Medical University of Graz
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Variable analysis
independent variables
- Genotype: WT, Siglec8+F+, Siglec8+F-, and SiglecF-/-
- Surface expression of Siglec-F, Siglec-8 and CCR3 on bone marrow cells throughout the differentiation process
- Cell viability (assessed with DAPI or Ghost fixable viability dye)
- Mature eosinophil morphology (Diff-Kwik staining and imaging)
- Bone marrow cells from the specified genotypes
- Differentiation protocol previously described by Dyer et al.
- Flow cytometry analysis as done previously
- Positive control: Not specified
- Negative control: Not specified
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