Every second, more than a million cells in the human body undergo a programmed form of self-destruction called apoptosis. This process is essential for everything from embryonic development to the routine turnover of aged cells. To prevent the accumulation of these dying cells, the body employs a disposal system known as efferocytosis, where phagocytes engulf the cellular remains. When dying cells are removed promptly, it prevents them from breaking down and releasing internal contents that could trigger inflammation, making this an immunologically quiet event foundational to tissue repair.
The Purpose of an Efferocytosis Assay
Scientists use an efferocytosis assay to measure the efficiency of this cellular cleanup process in a controlled setting. The assay allows researchers to observe how effectively phagocytes find, engulf, and process apoptotic cells. It provides a way to study the molecular signaling that occurs between the dying cell and the phagocyte.
The primary output of these experiments is a numerical value, such as a “phagocytic index” or “efferocytosis index.” This score represents the capacity of the phagocytes by indicating the average number of apoptotic cells consumed per phagocyte. It provides a standardized measure, allowing for comparisons across different conditions, like between healthy and diseased cells.
These tests investigate the rate and capacity of efferocytosis, determining if a genetic mutation or drug enhances or impairs the function. By isolating the process, researchers can dissect the specific molecular steps involved. This includes the “find-me” signals released by apoptotic cells and the “eat-me” signals on their surface that trigger engulfment.
Core Components and Procedure
A typical efferocytosis assay involves three main components: the phagocytes, the apoptotic target cells, and their co-culture. The “eater” cells are professional phagocytes like macrophages or dendritic cells. These can be sourced from cell lines or primary cells, like bone marrow-derived macrophages (BMDMs), cultured in the lab. The phagocytes are plated in a culture dish where they adhere and are prepared for the experiment.
The second component is the population of apoptotic target cells, often immortalized cell lines like Jurkat T cells. To induce apoptosis, these cells are treated with a stimulus like ultraviolet (UV) light or chemicals like staurosporine. This treatment causes the cells to display “eat-me” signals, most notably exposing the lipid phosphatidylserine on their outer surface.
To track their fate, apoptotic cells are labeled with a fluorescent dye, such as CFSE or Calcein AM. Once the phagocytes are prepared and the target cells are apoptotic and labeled, the two populations are mixed. This co-culture is incubated for a period, ranging from 30 minutes to a few hours, allowing efferocytosis to occur.
Common Measurement Techniques
Two primary technologies are used to measure efferocytosis: flow cytometry and fluorescence microscopy. Flow cytometry is a high-throughput technique that rapidly analyzes thousands of individual cells as they pass one by one through a laser beam. In this method, phagocytes that have engulfed fluorescently labeled apoptotic cells will themselves become fluorescent. The flow cytometer can distinguish between phagocytes that have not eaten any targets and those that have, based on the fluorescent signal. This allows for the rapid counting of a large number of cells, providing a statistically robust efferocytosis index.
Fluorescence microscopy offers a more visual approach, allowing scientists to directly observe the cells and see the fluorescently labeled apoptotic cells inside the phagocytes. To accurately distinguish between cells that are fully engulfed and those merely stuck to the outside, a dual-staining technique is often employed. For example, apoptotic cells might be labeled internally with a green dye, while an antibody labels any external targets with a red dye. A phagocyte containing only green fluorescence is scored as a successful efferocytosis event. This method also allows for live-cell imaging, where the entire process can be recorded.
Applications in Research and Disease
Measuring efferocytosis has implications for understanding and treating many human diseases. Defective clearance of apoptotic cells is a feature of many autoimmune conditions, like systemic lupus erythematosus (SLE). In SLE, the failure to clear dead cells leads to their secondary breakdown, releasing self-antigens that trigger the immune system to attack the body’s own tissues. Assays help study why this process fails and test drugs that might restore function.
The assay is also applied to chronic inflammatory diseases. In atherosclerosis, the failure of macrophages to clear dead foam cells exacerbates plaque buildup in artery walls. This contributes to the growth of the necrotic core within the plaque, a hallmark of high-risk lesions. Researchers use these assays to investigate dysregulated molecular signals and screen for compounds that could enhance cellular cleanup.
The assay is valuable in cancer research, as tumors can manipulate efferocytosis to create an immune-suppressive environment. For example, tumor-associated macrophages can clear apoptotic cancer cells in a way that dampens anti-tumor immune responses. Researchers are exploring ways to block this process, using efferocytosis assays to screen for inhibitory molecules. The assay is a tool in drug discovery, helping identify therapeutics that modulate efferocytosis.