Ghost fixable viability dye

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).

Murine dorsal skin was stored 24 h in Tissue Storage Solution (Miltenyi Biotech) and digested using RPMI medium 5% Fetal Bovine Serum (FBS) with Antimycotic/Antibiotic containing Liberase TL (1.67 Wunsch unites/mL) and DNAse (500 μg/mL) rotating at 37°C for 2h followed by filter separation (100→70→30 μm). After a 5′ application at RT of red blood cell lysis buffer, single cells were resuspended in FACS buffer (DPBS containing 1% BSA) followed by surface staining for viable (Ghost Fixable Viability Dye; Tonbo) neutrophils (CD45⁺ (clone 30-F11; Biolegend), CD11b⁺ (clone M1/70; Biolegend), and Ly6G⁺ (clone 1A8; Biolegend)) positive cells. Cell acquisition was performed on a Bio-RAD ZE5 flow cytometer and data analyzed using FlowJo software (Treestar).