Technical advance: autofluorescence as a tool for myeloid cell analysis

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.