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Updated: Feb 16, 2026
Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung
Published on: January 31, 2025
FACS isolation of live mouse eosinophils at high purity via a protocol that does not target Siglec F
Wendy E Geslewitz1, Caroline M Percopo1, Helene F Rosenberg1
1Inflammation Immunobiology Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
Abstract:
Flow cytometry protocols designed to identify mouse eosinophils typically target Siglec F, an α-2,3-sialic acid binding transmembrane protein expressed universally on cells of this lineage. While a convenient target, antibody-mediated ligation of Siglec F induces eosinophil apoptosis, which limits its usefulness for isolations that are to be followed by functional and/or gene expression studies. We present here a method for FACS isolation which does not target Siglec F and likewise utilizes no antibodies targeting IL5Rα (CD125) or CCR3. Single cell suspensions are prepared from lungs of mice that were sensitized and challenged with Aspergillus fumigatus antigens; eosinophils were identified and isolated by FACS as live SSChi/FSChi CD11c-Gr1-/loMHCII- cells. This strategy was also effective for eosinophil isolation from the lungs of IL5tg mice. Purity by visual inspection of stained cytospin preparations and by Siglec F-diagnostic flow cytometry was 98-99% and 97-99%, respectively. Eosinophils isolated by this method (yield, ~4×106/mouse) generated high-quality RNA suitable for gene expression analysis.
Insights
This study introduces a novel flow cytometry method for isolating mouse eosinophils without inducing cell death. This technique enables functional and gene expression studies by avoiding Siglec F antibody targeting.
Area of Science:
- Immunology
- Cell Biology
- Respiratory Medicine
Background:
- Traditional flow cytometry for mouse eosinophil identification targets Siglec F.
- Antibody-mediated Siglec F ligation causes eosinophil apoptosis, hindering downstream functional and gene expression analyses.
- Existing methods often rely on antibodies for IL5Rα (CD125) or CCR3, which may not be universally applicable.
Purpose of the Study:
- To develop a novel Fluorescence-Activated Cell Sorting (FACS) method for isolating mouse eosinophils.
- To enable functional and gene expression studies by avoiding apoptosis-inducing markers.
- To provide an alternative isolation strategy for eosinophils from mouse lungs.
Main Methods:
- Single cell suspensions were prepared from the lungs of mice sensitized and challenged with Aspergillus fumigatus antigens.
- Eosinophils were identified and isolated by FACS as live SSChi/FSChi CD11c-Gr1-/loMHCII- cells.
- The method was validated in IL5tg mice and assessed for purity and RNA quality.
Main Results:
- The developed FACS strategy successfully isolated eosinophils without targeting Siglec F, IL5Rα (CD125), or CCR3.
- Purity of isolated eosinophils was high, reaching 98-99% by cytospin and 97-99% by Siglec F flow cytometry.
- Isolated eosinophils yielded high-quality RNA suitable for gene expression analysis, with approximately 4×106 cells obtained per mouse.
Conclusions:
- A novel, non-apoptotic FACS method for mouse eosinophil isolation has been established.
- This method overcomes limitations of Siglec F-targeting protocols, facilitating subsequent functional and molecular studies.
- The technique is effective in models of allergic airway inflammation and in IL5tg mice, providing a valuable tool for respiratory immunology research.
