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.
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.
Real-time visualization and fully automated analysis of phagocytosis
A simple three step protocol devoid of wash steps or stopping of phagocytosis.
High sensitivity, requiring low phagocyte numbers
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.
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).
Treat cells—Activator/inhibitor or molecular intervention. Add the desired treatments (25 µL/well) at 3x final assay concentrations.
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).
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.
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 3x final assay concentration.
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.
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).
This protocol specifically describes the use of IncuCyte ZOOM® for monitoring bioparticle uptake in the presence of inhibitory agents in a single 96-well plate.
The following protocol is a detailed worked example designed to enable the user to run a successful phagocytosis assay without the need for seeding validation studies. It specifically describes the use of IncuCyte ZOOM® for monitoring bioparticle uptake in the presence of inhibitory agents in a single 96-well plate. In this example, mouse macrophage cell line J774A.1 is used.
Remove a freshly confluent (~80%) T75 flask of J774A.1 cells from the tissue culture incubator and place within a sterile cell culture hood.
Remove media and wash cells with D-PBS (10ml).
Add 10 ml fresh cell culture media to flask and detach cells by scraping.
Perform a cell count (e.g. trypan blue staining + haemocytometer).
Dilute the cell suspension to 2 x105 cells/ml in culture media.
Using a multi-channel pipette seed cells (50 µl per well, i.e. 1 x104 cells per well) into chosen wells of 96-well microplate.
Exclude cells from at least 3 wells of the microtitre plate. These will serve as a negative control (- cells/+ bioparticles). To these wells add 50 µl media instead of cell suspension.
Remove bubbles from all wells by gently squeezing a wash bottle (containing 100% ethanol with the inner straw removed) to blow vapor over the surface of each well. Keep the tip of the wash bottle approximately 5 cm from the media surface.
Allow the cells to settle in the hood for 30 minutes before placing in incubator overnight.
In the IncuCyte ZOOM® software schedule 24 hour repeat scans every 15 minutes.
Prepare dilutions of inhibitory agents in assay media at 3x final assay concentration and warm by placing in a 37°C incubator.
Remove bubbles from all wells.
Remove the cell plate and compounds from the incubator and to each well add 25 µl of the desired compound.
Replace cells in the incubator for desired incubation period.
Prepare a suspension of IncuCyte™ pHrodo® Bioparticles®:
Thoroughly mix the bioparticle suspension by vortexing and dilute bioparticles to 4x final assay concentration in cell media. Note the recommended final assay concentration is 10 µg per well.
Immediately after dilution of the bioparticles, remove the cell plate from the incubator and add the bioparticle suspension to the cell plate using a multichannel pipette. Add bioparticles to the control wells which contain no cells.
Exclude bioparticles from at least 3 wells of the plate which contain cells. This will serve as negative control (+ cells/- bioparticles). To these wells add 25 µl media instead of bioparticle suspension.
Remove bubbles from all wells and place cells in IncuCyte ZOOM®.
In the IncuCyte ZOOM® set scans to commence immediately and monitor increase in fluorescence.
J774A.1 murine macrophage cell line were seeded at 1 x104 cells/well and incubated with IncuCyte™ pHrodo® Green E. coli Bioparticles® (10 μg/well). Exemplar data show blended images of phase contrast and green fluorescent images (top row), fluorescent images alone (middle row) at 0, 1 and 4 hours incubation time.
Bottom row shows the masked fluorescent area. Briefly, the IncuCyte ZOOMTM software enables users to define a minimum fluorescence intensity threshold. Any fluorescent pixel which has an intensity above this threshold is considered to be above the signal-to-noise ratio and becomes ‘masked’. This masked area increases as more phagosomes engulf bioparticles and become fluorescent. Hence the metric ‘Total fluorescent object area’ is a useful surrogate for phagosome counting.
Above plot shows the increase in masked fluorescent object area (proportional to the number of phagosomes which have engulfed bioparticles). Fluorescence area and intensity increase where cells have been exposed to bioparticles (dark blue markers). In the absence of phagocytic cells (pale blue markers) no increase in fluorescence is observed.
As the phagosomes engulf bioparticles and become mature phagolysosomes, their pH continues to decrease. As this occurs the fluorescence intensity of the probe continues to increase.
Above plot shows the increase in intensity of fluorescent objects. This metric enables users to not only measure how many phagosomes have taken up bioparticles, but also measure their maturation.
Over time the number of phagocytosing cells increases, and cell proliferation can be quantified using the metric ‘Phase Object Confluence’: a measure of the area of field of view covered by cells.
To generate this metrics the user must create a Processing Definition suited to the cell type, assay conditions and magnification and then apply the Processing Definition to the data set as an Analysis Job.
On addition to the cells, bioparticles will be visible in phase images as fine granular material. In order to exclude these objects from the mask, use the Area Filter function when setting up the processing definition. By choosing a minimum area requirement, all particles defined as smaller than this area will be excluded from the mask.
The data can be exported into a third party data analysis program (e.g. Microsoft Excel) and increase in confluence plotted over time.
As the cells engulf IncuCyte™ pHrodo® Bioparticles®, the fluorescence intensity inside the cells increases. This can be reported in two ways: as an increase in fluorescence area (‘Total Object Area’); and as increase in intensity, integrated over the area of detectable fluorescence (‘Total Integrated Intensity’).
To generate these metrics the user must create a Processing Definition suited to the cell type, assay conditions and magnification and then apply the Processing Definition to the data set as an Analysis Job.
Follow the instruction provided within the ‘IncuCyte ZOOM® Fluorescent Processing Overview’ Technical Note to (1) create an appropriate image collection including the fluorescence channel with which to (2) create a Processing Definition to mask cells using fluorescent object analysis; (3) launch an Analysis Job to analyze images and produce metrics based on parameters contained within the saved Processing Definition.
To exclude background fluorescence from the mask, use the background subtraction feature in the Parameters drop-down menu. The feature ‘Top-Hat’ will subtract local background from brightly fluorescent objects within a given radius; this is a useful tool for analyzing objects which change in fluorescence intensity over time, as in this case.