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Phagocytosis of Bioparticles®

Incucyte® Phagocytosis Assays

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.

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Overview & video

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.

Real-time monitoring of phagocytosis using the IncuCyte® ZOOM live-cell imaging systemReal-time monitoring of phagocytosis using the IncuCyte® ZOOM live-cell imaging system

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.


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Application Note:

Gain additional insights into the mechanism of immune cell killing by combining live cell analysis and flow cytometry into a single workflow

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Assay Concept

  1. Macrophages (e.g. J774A.1 mouse macrophages) or other phagocytic cells are seeded in 96- or 384- well micro-titer plates.
  2. Phagocytosis is measured in real-time by adding mix-and-read sterile pHrodo® Bioparticles® for Incucyte® .
  3. An increase in cellular fluorescence indicates the internalization of pHrodo® Bioparticles® into the phagosome. Incucyte® automated image analysis enables quantitation of phagocytosis over time.

Phagocytosis Assay Concept

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

Time-lapse visualization of J774A

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

Quantification of phagocytosis using fluorescence segmentation

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

IncuCyte phagocytosis assay quick start guide

Incucyte® Phagocytosis Assay quick start guide.

View the general protocol


Highly sensitive, low background, low cell numbers

The magnitude of the response is cell number-dependent and Bioparticle® number-dependent.

Quantification of phagocytosis is cell number- and Bioparticle dependent

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.

Highly consistent phagocytosis signal with little or no background in the absence of cells or bioparticles

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® .

Inhibition of phagocytosis by Cytochalasin D, Latruculin A, Nocodazole

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
Real-time cell visualization
Integrated analysis
Mix-and-read, no wash or quench
Long-term kinetic measurements
Flexible choice of effector cells
No fixation/lifting
High sensitivity/low background
Low cell number/bioparticle number

Ordering Info

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 Product Data Sheet Safety Data Sheet  Qty. Catalog 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

Tech Resources

Technical Resources

Protocols

Posters

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

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