IncuCyte® Phagocytosis General Protocol

Overview

Phagocytosis is a specific form of endocytosis by which cells internalize solid matter, thus eliminating cellular debris and pathogens. While most cells are capable of phagocytosis, it is the phagocytes of the immune system, including macrophages, neutrophils and immature dendritic cells that specialize in this process. In these cells, phagocytosis is a mechanism by which micro-organisms can be contained, killed and processed for antigen presentation and represents a vital facet of the innate immune response to pathogens, and plays an essential role in initiating the adaptive immune response.

The process of phagocytosis begins with the binding of opsonins (complement or antibody) and/or pathogen-associated molecular pathogens (PAMPs), to cell surface receptors on the phagocyte. This causes receptor clustering and triggers phagocytosis. The cell membrane then extends around the target, eventually enveloping it to form a discreet phagosome. This vesicle matures and acidifies to form a phagolysosome, in which the contents degrade.

Common methods used to phagocytosis are usually end-point measurements, often complex and needing many wash/quench steps, require cell lifting (e.g. flow cytometry) and generally require the biology to be halted either chemically or thermally prior to reading. The particles to be phagocytosed have been traditionally conjugated to enzymes, antibodies or constitutively fluorescent probes. These approaches have the added complication of needing to remove or quench the probe to remove interference from non-engulfed material. More recently the introduction of pH-sensitive probes, such as pHrodo® conjugated reagents (IncuCyte® pHrodo® Bioparticles® for Phagocytosis) transition from a non-fluorescent state at neutral pH outside the phagocyte, to highly fluorescent within the low pH environment of the phagosome. Thus washing or quenching steps are no longer required, resulting in simplified protocols.

We have developed a new image-based methodology that combines IncuCyte® pHrodo® and image-based fluorescent measurements, enabling simple mix and read protocols suitable for screening.

The assay enables:

1. Real-time visualization and fully automated analysis of phagocytosis
2. A simple three step protocol devoid of wash steps or stopping of phagocytosis.
3. High sensitivity, requiring low phagocyte numbers

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Phagocytosis of bacterial or yeast bioparticles by macrophages

This protocol provides an overview of the IncuCyte® Phagocytosis Assay methodology. It is compatible with the IncuCyte ZOOM® instrument using your choice of phagocyte cells, in combination with IncuCyte® pHrodo® Bioparticles® for Phagocytosis reagents. Note that the mouse macrophage cell line J774A.1 was used to optimize the described conditions, however the methodology can be adapted to accommodate any phagocyte.

IncuCyte® Phagocytosis Assay concept. Phagocytes are combined with IncuCyte® pHrodo® Bioparticles® in the presence or absence of modulating treatments whilst being imaged within your incubator using the IncuCyte® live-cell imaging system. As the IncuCyte® pHrodo® Bioparticles® reach the acidic phagosome the fluorescence intensity of the fluorescent label is greatly enhanced, enabling phagocytosis to be quantified directly and in real-time.

1. Seed target cells—Phagocyte Cell Seeding. Seed phagocytes (50 µL/well, 1 x10³ to 1 x10⁴ cells/well) into the 96-well plate and leave to adhere (2 - 16 h).
2. Treat cells—Activator/inhibitor or molecular intervention. Add the desired treatments (25 µL/well) at 3x final assay concentrations.
3. Add IncuCyte® pHrodo® Bioparticles® for Phagocytosis—IncuCyte® pHrodo® Bioparticles® addition. Add your choice of Bioparticle® (e.g. E. coli, S. aureus, Zymosan) to the 96-well plate (approximately 10 µg per well depending on Bioparticle; 25 µL/well at 4x final assay concentrations).

General Protocol

Day 0:

1. Seed phagocytic cells (50 µL per well) at an appropriate density into a 96-well flat bottom plate (Corning, 3595) such that by day 1 the cell confluence is approximately 10 - 20%. The seeding density will need to be optimized for cell type used; however we have found that 1 x10³ to 1 x10⁴ cells per well are reasonable starting points.

• Phagocyte cell growth can be monitored by recording phase images using the IncuCyte® ZOOM live cell imaging device and confluence algorithm.

Day 1:

1. Once the target cells have reached appropriate confluence remove the cell plate from the incubator and add desired treatments. The volumes/dilutions may be varied; however we recommended 25 μL, prepared at 4x final assay concentration.

a. Incubate the treatments for the desired duration.

2. Prepare IncuCyte® pHrodo® Bioparticles® by resuspending to 1 mg/mL in PBS or complete media of choice. Transfer this solution to a glass vial, vortex and sonicate for a minimum of 5 minutes (longer sonication may be required for Zymosan).

a. Note the formation of a homogeneous suspension may be improved by initial reconstitution in PBS, followed by subsequent dilution in assay media (PBS final assay concentration of 5%).

3. After incubation with the treatments, add the IncuCyte® pHrodo® Bioparticles® of your choice to the plate; we recommend 10 μg per well for E. coli/S. aureus or 5 μg for Zymosan.

a. Remove bubbles at the liquid surface by gently squeezing a wash bottle (containing 100% ethanol with the inner straw removed) to blow vapor over the surface of each well.

4. Image the plate in the IncuCyte® ZOOM instrument with a 20x or 10x objective using the Standard Scan Type. We recommend 2 images per well, and scanning of phase and fluorescence every 15 minutes for 2-48 h (or until the fluorescence area and intensity plateaus).