Protocol detail

Eosinophil Identification Protocol

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Cells (3×10⁵) were incubated first with mouse FcBlock (2.4G2; BD PharMingen, San Diego, CA, USA) to inhibit nonspecific binding of antibodies. After washing, cells were stained with anti-Siglec F, anti-Gr-1, anti-CD11b, anti-CD11c and respective isotype controls (BD Biosciences, PharMingen). Numbers of positive cells were quantified by flow cytometry (FACSCanto flow cytometer, BD Biosciences, San Jose, CA). Eosinophils were identified as SiglecF⁺Gr-1⁺CD11b⁺CD11c^-[21] (link), [38] (link). Data were collected on a FACSCanto flow cytometer and analysed with FlowJo software (version 10, Tree Star, Inc).

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Yang M., Eyers F., Xiang Y., Guo M., Young I.G., Rosenberg H.F, & Foster P.S. (2014). Expression Profiling of Differentiating Eosinophils in Bone Marrow Cultures Predicts Functional Links between MicroRNAs and Their Target mRNAs. PLoS ONE, 9(5), e97537.

Publication 2014

anti-Siglec F anti-Gr-1 anti-CD11b anti-CD11c FACSCanto flow cytometer

Antibodies Cd11b Cells Eosinophils Flow cytometry Inhibit Isotype Mouse Siglec Tree

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Variable analysis

independent variables

Mouse FcBlock (2.4G2; BD PharMingen, San Diego, CA, USA)

dependent variables

Numbers of positive cells quantified by flow cytometry (FACSCanto flow cytometer, BD Biosciences, San Jose, CA)

control variables

Respective isotype controls (BD Biosciences, PharMingen)

controls

Positive control: Mouse FcBlock (2.4G2; BD PharMingen, San Diego, CA, USA) to inhibit nonspecific binding of antibodies
Negative control: Respective isotype controls (BD Biosciences, PharMingen)

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