Phagocytosis of Bioparticles®
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.
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® 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.
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.
Quantification of phagocytosis using fluorescence segmentation. Incucyte® software enables accurate segmentation of the fluorescence image (pink mask) and minimizes the impact of background fluorescence.
Incucyte® Phagocytosis Assay quick start guide.
The magnitude of the response is cell number-dependent and Bioparticle® number-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, 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. J774A.1 murine macrophages phagocytosing pHrodo® Green E. coli Bioparticles® for Incucyte® in the presence of inhibitors.
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|
|Mix-and-read, no wash or quench|
|Long-term kinetic measurements|
|Flexible choice of effector cells|
|High sensitivity/low background|
|Low cell number/bioparticle number|
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|