Related Experiment Video
Updated: Jun 12, 2026
Intracellular Phosphoflow Cytometry of Acute Myeloid Leukemia Patient-Derived Xenotransplants
Published on: June 6, 2025
Technical advance: autofluorescence as a tool for myeloid cell analysis
Andrew J Mitchell1, Lydie C Pradel, Lionel Chasson
1Centre d'Immunologie de Marseille-Luminy, INSERM-CNRS-Université de La Méditerranée, Marseille, France. andrewm@med.usyd.edu.au
Insights
Autofluorescence (AF) in flow cytometry reveals distinct myeloid cell populations, including resident monocytes and red blood cell-phagocytosing macrophages (RPM). This method enables purification of iron-rich RPM for deeper biological insights.
Area of Science:
- Immunology
- Cell Biology
- Biomedical Engineering
Background:
- Cellular autofluorescence (AF) is typically viewed as an obstacle in flow cytometric analysis.
- Conventional flow cytometry often struggles to resolve complex leukocyte subsets.
- Understanding myeloid cell heterogeneity is crucial for immunological research.
Purpose of the Study:
- To integrate cellular AF into flow cytometric analysis for improved leukocyte characterization.
- To identify and isolate distinct myeloid cell populations, particularly in complex biological samples.
- To leverage AF patterns for functional and metabolic profiling of immune cells.
Main Methods:
- Utilized a mouse model to examine cellular AF across various excitation and emission wavelengths.
- Employed gating strategies based on discrete AF patterns to isolate cell populations.
- Conducted surface marker expression analysis and gene expression profiling on isolated populations.
Main Results:
- Successfully identified major myeloid populations in the spleen, including resident monocytes and red blood cell-phagocytosing macrophages (RPM).
- RPM displayed a phenotype indicative of lipid and iron metabolism, with significant intracellular ferric iron content.
- Developed a magnetic-based purification method for iron-rich RPM.
- Extended the AF-based analysis to leukocytes from other organs, identifying previously elusive myeloid subsets.
Conclusions:
- Incorporating AF into flow cytometry provides a powerful tool for dissecting complex immune cell mixtures.
- AF analysis facilitates the simultaneous characterization and isolation of functionally distinct myeloid populations.
- This approach enhances the identification of biologically significant cell subsets beyond conventional methods.
Abstract:
Cellular AF is usually considered a hindrance to flow cytometric analysis. Here, we incorporate AF into analysis of complex mixtures of leukocytes. Using a mouse model, we examined cellular AF at multiple excitation and emission wavelengths, and populations with discrete patterns were gated and examined for surface marker expression. In the spleen, all major myeloid populations were identified. In particular, the approach allowed simultaneous characterization of RPM and resident monocytes. When monocytes and RPM were compared, RPM exhibited a phenotype that was consistent with involvement in physiological processes, including expression of genes involved in lipid and iron metabolism. The presence of large amounts of stored ferric iron within RPM enabled purification of these cells using a magnetic-based approach. When adapted for use on leukocytes isolated from a range of other organs, incorporation of AF into analysis allowed identification and isolation of biologically important myeloid populations, including subsets that were not readily identifiable by conventional cytometric analysis.
