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Quantification of Phagocytosis of Apoptotic Cells Using Flow Cytometry
Presenter: Mallary C. Greenlee
Mentor: Dr. Andrea Tenner
The innate immune system consists of humoral components, such as complement, and cellular
mechanisms, such as phagocytosis. This pathway directs the adaptive immune response. For example,
the innate immune system mediates clearance of apoptotic cells without initiating adaptive immunity,
a process which could lead to potentially detrimental inflammation. Humoral components known as
defense collagens, such as complement protein C1q, regulate phagocytic cellular responses, leading to
enhancement of phagocytic function, including the rapid clearance of apoptotic cells. We developed
a fluorescence-based phagocytosis assay to begin to explore defense collagen driven phagocytosis and
cellular activation. To generate apoptotic cells, Jurkat T cells were treated with etoposide. Apoptosis
was assessed by measurement of cell surface phosphatidylserine. Exclusion of vital dye 7-AAD was
also assessed to ensure the cells were not necrotic. In addition, PARP cleavage in apoptotic cells and
absence of cleavage in healthy and necrotic cells demonstrated specificity for apoptotic cell death.
Apoptotic and healthy jurkats labeled with the fluorescent dye, CFSE, were incubated with
phagocytic human monocyte derived macrophages. Phagocytosis was measured with flow cytometry
by detecting CD11b+ macrophages that also contained CFSE. We found that apoptotic cells were
preferentially engulfed by macrophages compared to engulfment of healthy cells. Furthermore,
presence of serum (containing defense collagens) enhanced phagocytosis. Currently, we are testing
the ability of purified C1q to bind to apoptotic Jurkats cells to continue to investigate the mechanism
by which C1q and other defense collagens regulate phagocytosis of apoptotic cells.