Phagocytosis of Cells & Efferocytosis
The phagocytosis of diseased or dying cells is a key function of phagocytic immune cells such as macrophages. The clearance of dead or apoptotic cells by phagocytosis is called efferocytosis and is critical to resolving episodes of inflammation (e.g. removal of neutrophils that persist following inflammation) and preventing the development of chronic inflammatory or autoimmune diseases (e.g., COPD, asthma, rheumatoid arthritis, fibrosis, and atherosclerosis). Efferocytosis is also key for the efficient removal of apoptotic tumour cells following treatment with anti-cancer drugs ensuring that cellular contents are contained and circumventing unwanted inflammatory responses.
Cancer cells need not die in order to be removed by phagocytic cells. Tumor cells can be targeted with agents that promote phagocytic uptake and clearance by labelling the cell with agents that engage phagocytic cells. Antibody dependent cellular phagocytosis (ADCP) is triggered by the addition of antibodies such as Rituximab (an anti-CD20 monoclonal antibody which targets B cells). A similar strategy is the use of antibodies that block expression of “don’t-eat-me” signals (e.g. CD47), a mechanism by which tumor cells evade clearance. Anti-CD47 antibodies block this signal allowing the cells to be recognized and removed by phagocytes.
Introducing the Incucyte® Cell Phagocytosis Assays
The Incucyte® Live-Cell Analysis System enables real-time, automated cellular phagocytosis and efferocytosis assays inside your cell culture incubator.
Automatically quantify the phagocytosis of cells over the entire assay time course. Make real-time measurements of cell internalization using your choice of target cells labeled with validated pH sensitive probes (Incucyte® pHrodo® Red Cell Labeling for Phagocytosis). Confirm phagocytosis signals using Incucyte® high definition phase contrast images to directly visualize cell engulfment.
- Screening compounds for inhibition/activation of efferocytosis
- Quantifying antibody mediated cell phagocytosis (e.g., anti-CD47 antibody mediated phagocytosis or ADCP)
Video. Real-time monitoring of phagocytosis of apoptotic cells using the Incucyte® live-cell analysis system. Time-lapse movies acquired using Incucyte® system showing real-time visualisation of Incucyte® pHrodo® Red Labeled apoptotic Jurkat T lymphocytes engulfed by the mouse macrophage cell type J774A.1. Apoptotic Jurkat cells are phagocytosed and, on entering the acidic environment of the phagosome, increase in fluorescence. Incucyte® integrated image analysis tools enable detection and measurement of the red fluorescent signal over the entire assay time-course.
Macrophages (e.g. J774A.1 mouse macrophages) or other phagocytic cells are seeded in 96- or 384- well micro-titer plates.
Target apoptotic cells (e.g., neutrophils, Jurkats) labeled using the Incucyte® pHrodo® Red Cell Labeling Kit for Phagocytosis are added and phagocytic uptake of the target cells is measured in real-time
An increase in cellular fluorescence indicates the internalization of target cells into the phagosome. Incucyte® automated image analysis enables quantitation of cell phagocytosis over time.
- Visualize and quantify phagocytosis of cells using time-lapse imaging
- Automated analysis over the entire assay time course
- Simple mix and read 96/384-well protocols, no washing, no fixing, no lifting
- Make relevant measurements in a highly flexible assay format
- Incucyte® pHrodo® Red Cell Labeling Kit compatible with your choice of phagocyte/target cells
Visualize and quantify phagocytosis using time-lapse imaging
- Observe and measure phagocytosis in real time using intuitive Incucyte® image analysis tools. Validate treatment effects with images and movies
- The pHrodo fluorescent signal is present only when target cells are internalized eliminating the risk of artefact signals
Figure 1. Visualize and validate the phagocytosis of cells (efferocytosis) with images and movies. Time-lapse images of J774A.1 mouse macrophages phagocytosing apoptotic Jurkats labeled using the Incucyte® pHrodo® Red Cell Labeling Kit over 24 hours. Correlate fluorescent signal with phenotypic changes associated with phagocytosis. Quantify efferocytosis using Incucyte® image analysis tools. User-friendly software enables direct image-based detection of phagocytosed apoptotic cells (blue mask).
Automated analysis over the entire assay time course
Determine how and when treatment effects occurred without removing cells from the stable environment of the incubator.
Figure 2. Quantify treatment effects automatically and non-invasively. Every well of a 96/384 well plate is imaged and analyzed automatically to provide a microplate readout of efferocytosis over time (left). Time-courses reveal concentration-dependent treatment effects (center). Transform data into concentration-response curves to compare pharmacology (right).
Simple 96/384-well protocols, no washing, no fixing, no lifting
Figure 3. Quick guide. 1) Seed phagocytotic cell (e.g. J774A.1, 1-10K/well) in 96-well plates. Culture overnight (50 µL/well). 2) Treat effector cells with compounds prior to phagocytosis (0.5-24 h pre-treatment, 25 µL/well). 3) Add Incucyte pHrodo-labeled target cells to treated wells (10 µL/well, 25 µL/well). 4) Capture images every 10-20 mins (20x or 10x) in an Incucyte system for 2-18 hours. Analyze using integrated software.
Make relevant measurements in a highly flexible assay format
- Use your choice of phagocytic and target cells to measure phagocytosis of diseased, dead or dying cells. Investigate antibody mediated cellular phagocytosis (e.g., anti-CD47 antibody mediated phagocytosis / ADCP) or efferocytosis (phagocytosis of apoptotic cells).
Figure 2. Flexible choice of target and effector cells. Viable T lymphoblast cells CCRF-CEM were labeled with the Incucyte® pHrodo® Red Cell Labeling Kit and exposed to either anti-CD47 monoclonal antibody or IgG isotype control. The anti-CD47 antibody binds to the 'don't eat me' signal on CCRF-CEM cells to promote macrophage mediated phagocytosis. In the presence of anti-CD47 target cells were engulfed by bone marrow-derived macrophages, increasing the fluorescent signal and producing a concentration-dependent effect. The presence of isotype control IgG had no effect on engulfment at all concentrations tested.
Phagocytosis of Cells FAQs
I need to characterize several different macrophage cell lines to find the one with the highest baseline rate of phagocytosis. How do I get started comparing macrophage phagocytosis across cell lines?
Comparing the phagocytic capabilities of different macrophage cell lines requires stable and reproducible culture conditions and frequent monitoring of phagocytic activity. A good starting place is to run head-to-head phagocytosis assays, using bait or target cells that have been treated with a pH-sensitive dye, and compare the number of phagosomes containing fluorescence at regular intervals. Assays that require frequent monitoring and imaging benefit from being conducted with a live-cell analysis platform, enabling regular imaging without perturbing the culture system.
Learn more about analyzing macrophage phagocytosis*** [LINK TO PHAGO BIOPARTICLES PAGE]
What is efferocytosis, and how does it differ from phagocytosis? Can I study neutrophil efferocytosis with a phagocytosis assay?
Efferocytosis is a term used to describe the phagocytosis of dead or apoptotic cells, as opposed to living cells, cell fragments or other debris. Therefore, phagocytosis assays can detect efferocytosis in neutrophils and any other phagocytic cell type.
Our lab uses a phagocytosis assay as a primary screen for induction of macrophage activation, but the method is highly end-user dependent. How can we standardize our phagocytosis assay and improve data reliability?
As with any assay based on live organisms, it's impossible to control for every influencing factor, but by standardizing your phagocytosis assay with in-incubator live-cell analysis you can cut out a major source of variability — the amount of time your plates are outside of the incubator for imaging. Live-cell analysis enables regular and comprehensive analysis of macrophage phagocytosis, without the hands-on component.
In phagocytosis assays, is there a size limit on the target cells for them to be engulfed by the phagocytic cells? Do they need to be below a specific size threshold?
In general, the speed and efficiency of phagocytosis is tied to both the size and the shape of the engulfed item, and a motivated phagocyte will attempt to engulf anything in its path. Unless your target cells are abnormally large or oddly shaped, there is no practical limit on cell size or cell shape when selecting the target cells for your phagocytosis assay.
Phagocytosis assays built entirely on visual observation would be appropriate for studying phagocytosis in macrophages with lysosomal insufficiency. However, since most other phagocytosis assays rely on the pH change that occurs upon phagosome fusion with endosomal compartments for color change, macrophages with lysosomal insufficiency may not provide meaningful data. The value of pH-sensitive indicators for macrophage phagocytosis in these cases should be experimentally determined.