What Is Phagocytosis?
Phagocytosis is a specific form of endocytosis by which cells engulf and internalize solid matter. Macrophages, neutrophils and immature dendritic cells are the major phagocytic cells of the immune system and specialize in this process. Phagocytosis is a key mechanism by which micro-organisms are contained, killed and processed for antigen presentation and represents a vital facet of the innate immune response to pathogens.
Application
Introducing the Incucyte® Phagocytosis Assay
A simple mix-and-read imaging assay for automated quantification of phagocytosis over time in living cells, directly within your incubator. The assay provides real-time visualization and analysis of the internalization of bioparticles using pH sensitive-conjugated probes, and is ideal for monitoring phagocytosis of bacterial Gram positive, Gram negative or yeast-derived pathogens by immune cells.
Videos. Real-time monitoring of phagocytosis using the Incucyte® Live-Cell Analysis System. Time-lapse movies acquired using Incucyte® live-cell imaging and analysis showing real-time visualization of pHrodo® Green E. coli Bioparticles® for Incucyte® engulfed by the mouse macrophage cell type J774A.1. The pHrodo® Bioparticles® 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 green fluorescent signal over the entire assay time-course and minimize the impact of background fluorescence. Note the dynamic activity of the macrophages and engulfment of bioparticles over time via the formation of phagocytic cups.
Assay Concept
- Macrophages (e.g. J774A.1 mouse macrophages) or other phagocytic cells are seeded in 96- or 384- well micro-titer plates.
- Phagocytosis is measured in real-time by adding mix-and-read sterile pHrodo® Bioparticles® for Incucyte®.
- An increase in cellular fluorescence indicates the internalization of pHrodo® Bioparticles® into the phagosome. Incucyte® automated image analysis enables quantitation of phagocytosis over time.
Key Advantages
Key Advantages of the Incucyte® Phagocytosis Assay
- Validation of phagocytosis using time-lapse imaging
- Automated analysis and quantification
- Mix-and-read 96/384-well format, no fixing, no quenching, no lifting
- Highly sensitive, low background, low cell numbers
- Sterile pHrodo® Bioparticles® for Incucyte® enable long-term measurements (0 to >48 hours) in real time
Validation of phagocytosis using time-lapse imaging
Figure 1. Time-lapse visualization of J774A.1 mouse macrophages phagocytosing pHrodo® Green E. coli Bioparticles® for Incucyte® over 4 hours. Images verify the presence of fluorescent punctate (phagosomal) labelled structures in the cytosol but not in the nucleus.
Automated analysis and quantification
Figure 2. Quantification of phagocytosis using fluorescence segmentation. Incucyte® software enables accurate segmentation of the fluorescence image (pink mask) and minimizes the impact of background fluorescence.
Mix-and-Read 96/384-well format - no fixing, no quenching, no lifting
Figure 3. Incucyte® Phagocytosis Assay quick start guide.
Highly sensitive, low background, low cell numbers
The magnitude of the response is cell number-dependent and Bioparticle® number-dependent.
Figure 4. Quantification of phagocytosis is cell number- and Bioparticle® dependent. J774A.1 mouse macrophages phagocytosing pHrodo® Green S. aureus Bioparticles® for Incucyte® (96-well format). Note high signal : background ratio with as low as 3K cells per well.
Figure 5. Highly consistent phagocytosis signal with little or no background in the absence of cells, Bioparticles® or inhibitor (cytochalasin D). Plateview illustrating the time-courses of phagocytosis by J774A.1 cells of pHrodo® Green E. coli Bioparticles® for Incucyte®.
Figure 6. Inhibition of phagocytosis by Cytochalasin D, Latruculin A, Nocodazole. J774A.1 murine macrophages phagocytosing pHrodo® Green E. coli Bioparticles® for Incucyte® in the presence of inhibitors.
Incucyte® Phagocytosis Assay vs. Other Common Approaches
Common methods used to assess phagocytosis are often single point (e.g. High Content Analysis (HCA)), require cell lifting (e.g. flow cytometry) or washing/quenching of the labelled pathogen (e.g. fluorescein labelled).
Incucyte® Live-Cell Analysis | Plate reader | Flow cytometry | HCA | ELISA | |
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Real-time cell visualization |
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Integrated analysis |
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Mix-and-read, no wash or quench |
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Long-term kinetic measurements |
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Flexible choice of effector cells |
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No fixation/lifting |
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High sensitivity/low background |
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Low cell number/bioparticle number |
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Ordering Information
Ordering Information
pHrodo® Bioparticles® for Incucyte® are fully validated for use with the Incucyte® Live-Cell Analysis System and are sterilized to enable long-term measurements of phagocytosis (0 to >48 hours).
Product | Qty. | Cat. No. |
|---|---|---|
pHrodo® Red E. coli Bioparticles® for Incucyte® | 2 mg | 4615 |
pHrodo® Green E. coli Bioparticles® for Incucyte® | 2 mg | 4616 |
pHrodo® Red Zymosan Bioparticles® for Incucyte® | 1 mg | 4617 |
pHrodo® Green Zymosan Bioparticles® for Incucyte® | 1 mg | 4618 |
pHrodo® Red S. aureus Bioparticles® for Incucyte® | 2 mg | 4619 |
pHrodo® Green S. aureus Bioparticles® for Incucyte® | 2 mg | 4620 |
Resources
Literature and Documentation
Related publications
A novel real time imaging platform to quantify macrophage phagocytosis
Researchers at The University of Oxford use Incucyte® live-cell analysis system to quantify phagocytosis with a higher degree of accuracy and sensitivity than existing methods. Incucyte® enables detailed in vitro investigation of primary phagocytes even when cell populations are rare or limited. Kapellos TS, Taylor L, Lee H, Cowley SA, James WS, Iqbal AJ, Greaves DR doi: 10.1016/j.bcp.2016.07.011