Phagocytosis of apoptotic cells by human macrophages: analysis by multiparameter flow cytometry.
BACKGROUND
Phagocytic removal of apoptotic cells is an important regulatory event in development, tissue homoeostasis, and inflammation. There are several methodologic problems with most in vitro studies of the molecular mechanisms of apoptotic cell phagocytosis. First, cell loss occurs during rigorous washing of adherent macrophages required to ensure removal of noningested particles. Second, discrimination of adherent or internalised apoptotic cells is difficult. Third, microscopic quantification is time consuming and has the potential for significant interobserver error. Fourth, subsequent analysis of phagocyte populations is difficult.


METHODS
We used a flow cytometric method that allows quantification of phagocytosis of fluorescently labelled apoptotic cells with the use of multiparameter flow cytometric analysis.


RESULTS
Phagocytosis of apoptotic cells was validated by use of inhibitors (cytochalasins) or low temperature and counterstaining with cell surface markers for the phagocytic targets to exclude binding to the phagocytic surface. Populations of phagocytic macrophages were sorted, and the presence of internalized apoptotic material was validated by microscopy.


CONCLUSIONS
The technique we used in this study allows observer-independent analysis of phagocytosis of apoptotic cells by macrophages. Importantly, phagocytic or nonphagocytic populations could be subjected to further characterization with the use of flow cytometry with additional fluorochrome reagents and can be re-cultured to study underlying regulatory mechanisms.
Jersmann
Hubertus P A
HP
MRC Centre for Inflammation Research, University of Edinburgh Medical School, Edinburgh, United Kingdom.
Ross
Katherine A
KA
Vivers
Sharon
S
Brown
Simon B
SB
Haslett
Christopher
C
Dransfield
Ian
I
D000954:Antigens, Surface / Q000276:immunology; D017209:Apoptosis / Q000502:physiology*; D003572:Cytochalasins / Q000494:pharmacology; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000502:physiology*; D009504:Neutrophils / Q000166:cytology / Q000502:physiology; D010587:Phagocytosis / Q000502:physiology*; D015203:Reproducibility of Results
D000954:Antigens, Surface; D003572:Cytochalasins; D005452:Fluoresceins; D005456:Fluorescent Dyes; C069306:5-chloromethylfluorescein