Technical advance: autofluorescence as a tool for myeloid cell analysis.
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.
Mitchell
Andrew J
AJ
Centre d'Immunologie de Marseille-Luminy, INSERM-CNRS-Université de La Méditerranée, Marseille, France. andrewm@med.usyd.edu.au
Pradel
Lydie C
LC
Chasson
Lionel
L
Van Rooijen
Nico
N
Grau
Georges E
GE
Hunt
Nicholas H
NH
Chimini
Giovanna
G
D000818:Animals; D002469:Cell Separation; D004002:Clodronic Acid / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005786:Gene Expression Regulation / Q000187:drug effects; D008081:Liposomes / Q000378:metabolism; D008099:Liver / Q000166:cytology; D008168:Lung / Q000166:cytology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D009000:Monocytes / Q000166:cytology / Q000187:drug effects; D022423:Myeloid Cells / Q000166:cytology* / Q000187:drug effects; D009928:Organ Specificity / Q000187:drug effects; D010586:Phagocytes / Q000166:cytology / Q000187:drug effects; D013154:Spleen / Q000166:cytology
D008081:Liposomes; D004002:Clodronic Acid