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METHODS IN CELL PHYSIOLOGY
1Program in Cell Biology, Hospital for Sick Children, and 2Institute of Medical Science and 3Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
Submitted 28 June 2006 ; accepted in final form 26 September 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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receptor-mediated phagocytosis of IgG-opsonized particles by cultured murine macrophages. Phagosomal acidification was measured as an index of maturation. The same platform was similarly used to implement high-throughput assays of invasion of mammalian cells by pathogenic bacteria. The invasion of HeLa cells by Salmonella and the subsequent intracellular proliferation of the bacteria were measured rapidly and reliably in large populations of cells. These high-throughput methods are ideally suited for the efficient screening of chemical libraries to select potential drugs and of small interference RNA libraries to identify essential molecules involved in critical steps of the immune response. Salmonella; phosphatidylinositol 3-kinase; actin; vacuolar pH
receptors that bind to the constant region of IgG are among the most studied and best understood (1). When exposed to particles coated with IgG, Fc receptors undergo clustering on the surface of the phagocyte, triggering signals that culminate in pseudopod extension and engulfment of the particles into membrane-bound vacuoles known as phagosomes (1, 2). The limiting membrane of the phagosome and its contents undergo a rapid and extensive remodeling, giving rise to a highly microbicidal organelle that mediates the destruction of the pathogen (4). The complex fusion and fission events that underlie remodeling are collectively known as phagosomal maturation. A number of human pathogens evade the microbicidal response of the innate immune system by impairing phagocytosis or by precluding phagosomal remodeling. Several genera of bacteria, such as Salmonella and Shigella, actively seek to enter mammalian cells, where they survive by coopting the host cell signaling and trafficking machinery (for review see Ref. 16). This is often accomplished through the delivery of bacterial effector proteins into the cytoplasm of the host cell by a specialized secretion apparatus (8). The intracellular niche provides the pathogen not only with a source of nutrients but also refuge from immune sentinels and from circulating antimicrobial agents.
Progress in the fight against infectious pathogens will require an improved understanding of their biology in the context of host cells and of the molecular basis of the immune response. Current experimental paradigms used to study phagocytosis, phagosome maturation, and bacterial invasion involve labor-intensive microscopic techniques. Each particle or organelle must be individually observed and scored; this is a time-consuming process that limits the number of experimental conditions that can be examined. Such approaches are not compatible with the efficient analysis of chemical libraries to screen for potential drugs or of small interference RNA libraries to identify essential molecules involved in host-pathogen interactions.
To circumvent some of these limitations, we have implemented and optimized an automated method for the study of phagocytosis, phagosome maturation, and bacterial invasion. The method, based on the recent development of robotic, high-throughput fluorescence microscopes, enables investigators to perform rapid analyses of particle engulfment and/or phagosome remodeling in large numbers of samples. Essential aspects of the biological preparations, hardware, and software required for such measurements, as well as information on potential applications of the system, are presented to provide a better understanding of the capabilities and limitations of high-throughput microscopy.
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MATERIALS AND METHODS |
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Reagents. Rabbit polyclonal antibodies to Salmonella were purchased from BD Biosciences (San Jose, CA); Cy2-, Cy3-, and Cy5-conjugated donkey anti-human and Cy2-conjugated anti-rabbit secondary antibodies from Jackson ImmunoResearch Laboratories (West Grove, PA); cytochalasin D (CytD) and LY-294002 from Calbiochem (San Diego, CA); 3.1- and 8.3-µm-diameter styrene-divinylbenzene copolymer (latex) beads from Bangs Laboratories (Fishers, IN); and Hoechst 33342, Cell Tracker Green, and LysoTracker Red from Molecular Probes (Eugene, OR). Human IgG and all other reagents were purchased from Sigma-Aldrich Canada (Mississauga, ON, Canada) unless otherwise specified.
Phagocytosis of beads. RAW264.7 cells were seeded in Costar 24-well plates (Corning, Acton, MA) on the night before the experiment. Latex beads were opsonized with human IgG (1.67 mg/ml) for 2 h at room temperature and washed three times in PBS. The beads were then prelabeled with Cy2- or Cy3-conjugated donkey anti-human secondary antibody (1:100 dilution) for 1–4 h at room temperature, washed three times, and resuspended in 500 µl of PBS. Where specified, the cells were pretreated with 10 µM CytD for 15 min or 100 µM LY-294002 for 30 min. A 15-µl aliquot of the bead suspension was added to each well, and the plate was centrifuged at 300 g for 1 min to deposit the beads onto the cells, thereby synchronizing the initiation of phagocytosis. Then cells were incubated at 37°C for 15 min to allow for bead internalization. The cells were placed on ice and washed with cold DMEM to remove uninternalized beads. The remaining external beads were stained by addition of a Cy2- or Cy5-conjugated donkey anti-human antibody (1:500 dilution) for 7.5 min in the cold and washed extensively.
For phagocytic index assays, cells were fixed with 4% paraformaldehyde in PBS for 20 min and washed with 100 mM glycine for 10 min. Cell nuclei were stained with Hoechst 33342 (8 µg/ml) for 10 min, washed once, and maintained in PBS. Where indicated, cells were prelabeled with Cell Tracker Green (2 µg/ml) for 30 min at 37°C and then washed three times with PBS before the addition of the latex beads.
For phagosome maturation experiments, phagocytosis was initiated, and, after 60 min, the cells were washed with cold DMEM and placed on ice to arrest maturation. External beads were labeled with Cy5-conjugated anti-human antibody (1:500 dilution) for 7.5 min and washed three times. Acidic intracellular compartments were labeled by incubation with 1 µM LysoTracker Red for 1 min. Cells were washed with cold PBS and brought directly to the high-throughput microscope for imaging. The imaging chamber was maintained at room temperature.
Bacterial invasion. HeLa cells were plated on 24-well plates on the night before the experiment. One milliliter of Luria-Bertani broth containing ampicillin (100 µg/ml) was inoculated with 30 µl of an overnight culture of Salmonella typhimurium and grown to late-log phase (3 h at 37°C with vigorous shaking). The bacteria were washed once and resuspended in 1 ml of PBS before use. HeLa cells were washed once and placed in serum-free DMEM before addition of a 5-µl inoculum of bacterial culture to each well. Bacterial invasion was allowed to proceed for 15 min at 37°C. The cells were washed three times to remove the majority of extracellular bacteria and then fixed as described above. The remaining extracellular bacteria were stained using rabbit anti-Salmonella antibodies (1:250 dilution) for 45 min followed by a Cy2-conjugated donkey anti-rabbit secondary antibody. Nuclei were subsequently labeled with Hoechst 33342 (8 µg/ml) for 10 min at room temperature. Where indicated, cells were pretreated with 10 µM CytD for 15 min or 100 µM LY-294002 for 30 min before addition of bacteria.
Intracellular bacterial proliferation assays were carried out as described above by fixing infected cells at the indicated times after bacterial invasion. After 30 min of proliferation, gentamicin (50 µg/ml) was added to the unfixed wells to kill external bacteria. For long incubation times, the concentration of gentamicin was decreased after 3 h to 5 µg/ml. Any remaining extracellular bacteria were labeled with rabbit anti-Salmonella antibody (1:250 dilution) for 45 min followed by Cy2-conjugated donkey anti-rabbit secondary antibody (1:1,000 dilution) for 45 min. Nuclei were stained with Hoechst 33342 as described above; the fixed cells were washed with PBS and used for image acquisition.
Image acquisition. Images were acquired using the KineticScan HCS Reader (Cellomics, Pittsburgh, PA), an integrated system capable of automated acquisition and analysis of large populations of individual cells, following the manufacturer's instructions. Briefly, the 24-well plates were placed in a temperature-controlled chamber maintained at room temperature or 37°C under 5% CO2. Images were acquired using the XF93 filter set (Omega Optical, Brattleboro, VT) and x20 or x40 objectives (0.4 and 0.5 NA, respectively). The microscope was set to autofocus every two fields.
Image analysis. The Cellomics software uses an object-identification algorithm based on intensity thresholds between adjacent pixels. Identified objects within an imaged field can subsequently be accepted or rejected for analysis on the basis of object characteristics, such as area, shape, and fluorescent intensity. Once a primary object is identified and accepted as valid, an area around (or within) the object can be defined for use in the subsequent quantitation procedure. The analysis of the determined regions of interest is refined by prepackaged generic and adaptable image analysis software modules termed BioApplications.
To determine the phagocytic index, we used Spot Detector BioApplication (version 2, Cellomics) software. Objects (i.e., individual cells) were identified by their stained nuclei, and a region delineating 35 pixels (corresponding to 
20 µm) beyond the edge of the nucleus was defined to encompass the perimeter of the cell and all cell-associated beads. The number of distinct spots within this boundary was calculated for total (Cy3-labeled) and external (Cy2-labeled) beads. Similar to the primary object identification protocol, the Spot Detector BioApplication selects spots within the target region on the basis of user-defined parameters. The number of internalized beads was calculated by subtraction of the number of external beads from the total number of beads. In turn, the phagocytic index was computed by dividing the number of internalized beads by the number of cells. Similarly, index of invasion was calculated as the number of internalized bacteria (total bacteria – external bacteria) per cell.
For quantitation of object fluorescence intensity levels, the Compartmental Analysis BioApplication (version 1, Cellomics) was used. Prelabeled fluorescent beads were identified as the primary object, with a circular region of interest extending two pixels (1 µm) beyond the edge of the bead undergoing analysis. Total fluorescence intensity within the region was determined for each fluorophore of interest. To further refine the analysis, the subpopulation of external beads was excluded from the analysis through identification using their Cy5 fluorescence. The remaining beads were considered to be internalized and were used for further analysis of phagosome acidification. A similar approach was used to determine intracellular bacterial proliferation.
Data analysis was performed using Microsoft Excel, and, where necessary, data were imported into FlowJo (Tree Star, Ashland, OR) using Text2FCS (ftp://facs.scripps.edu/pub/pc/txt2fcs.exe; Joseph Trotter, Scripps Research Institute, La Jolla, CA). Images and figures were prepared using Adobe Photoshop CS and Adobe Illustrator CS software (Adobe Systems, San Jose, CA).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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receptor-mediated phagocytosis employing RAW264.7 macrophages, hereafter called RAW cells. Accurate quantification of the phagocytic index necessitates correct identification of cells that have internalized opsonized particles. In principle, phagocytic cells can be identified using a whole cell (cytosolic) marker or a nuclear stain. To ensure that both methods yield comparable results, RAW cells were dual labeled with a nuclear stain (Hoechst 33342) and the cytoplasmic marker Cell Tracker Green before they were imaged with the Cellomics KineticScan HCS Reader. The nuclear (Fig. 1A) and cytoplasmic (Fig. 1B) channels for the same 50 fields were sequentially visualized by an automated imaging system, and the images were analyzed using object-recognition algorithms that discriminate cells on the basis of the contrast between adjacent pixels. Subsequently, identified objects were accepted or rejected as valid by criteria such as intensity and size. This analysis algorithm identified 1,554 and 1,631 valid objects in the Hoechst 33342 and Cell Tracker channels, respectively (Fig. 1D), confirming the ability of both approaches to accurately identify cells. Best-fitting regions delineating 35 pixels (corresponding to 20 µm) beyond the edge of accepted nuclei were defined for further analysis and are shown in orange (Fig. 1C).
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Actin polymerization and phosphatidylinositol 3-kinase in phagocytosis.
Actin polymerization and phosphatidylinositol 3-kinase (PI3K) activity are essential for Fc receptor-mediated phagocytosis (2, 12, 15, 21). To further validate our assay, we examined the effect of CytD, a potent inhibitor of actin remodeling (14), and LY-294002, a competitive inhibitor of PI3K (20), on the phagocytosis of IgG-opsonized beads. RAW cells were exposed to 3.1- or 8.3-µm-diameter Cy3-labeled opsonized latex beads for 15 min and then incubated with Cy5-conjugated anti-human antibodies to identify external beads. After extensive washing, the cells were fixed, and the nuclei were labeled with Hoechst 33342 and imaged. Where indicated, cells were pretreated with 10 µM CytD or 100 µM LY-294002 for 15 and 30 min, respectively. After acquisition of 50 fields for each condition, image analysis was performed using the Compartmental Analysis BioApplication. Individual beads were identified as the primary object by their Cy3 labeling, and their Cy3 and Cy5 fluorescence intensities were computed and analyzed. Figure 2, A–C, shows dot plots of the Cy3 and Cy5 fluorescence of the untreated and CytD- and LY-294002-treated 3.1-µm-diameter latex beads. In all three conditions, there are two populations of beads: internalized beads with Cy3, but no Cy5, staining (along the abscissa) and external beads with both labels (along the diagonal). Both inhibitors reduced the number of cell-associated internalized beads, indicating inhibition of phagocytosis and confirming the validity of our automated assay (Fig. 2, A–C).
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PI3K dependence of phagosome maturation. The phagocytic index assays described above indicate that this high-throughput system can accurately quantify particle internalization on a large scale. The system can also be employed to examine phagosome maturation. Because phagosomes become endowed with V-ATPases as they fuse with components of the endocytic pathway, measurement of their luminal pH has been used to monitor this maturation process (5, 18). Therefore, we employed the acidotropic dye LysoTracker Red to study phagosome maturation in a high-throughput fashion. RAW cells were allowed to internalize Cy2-labeled latex beads (Fig. 3A). After 60 min of phagocytosis, the cells were placed on ice to arrest maturation, and external beads were identified with Cy5-conjugated anti-human antibodies (Fig. 3C). Acidic compartments were then labeled by incubation with 1 µM LysoTracker Red (Fig. 3B). The live cells were washed with cold PBS and used immediately for image acquisition. Internalized beads show lower Cy2 fluorescence intensity than external beads (Fig. 3A, insets 1 and 2), possibly due to proteolysis of the labeling antibodies and/or quenching of fluorescence by phagosomal constituents. Phagosomes containing internalized beads (positive for Cy2 but devoid of Cy5) readily accumulated LysoTracker Red, indicating that they had undergone a considerable acidification. Accordingly, a dot plot (Fig. 3D) revealed that LysoTracker Red accumulated only around beads with negligible Cy5 fluorescence, whereas Cy5-stained (external) beads were not associated with the acidotropic dye. A histogram generated after gating separately the internal [Cy5-negative (G1)] and external [Cy5-positive (G2)] beads indicates that phagosomal acidification can be readily detected (Fig. 3E). Accumulation of LysoTracker Red in response to phagosomal acidification was verified using a weak base. Cells were exposed to 10 mM NH4Cl in PBS 1 min before and during application of LysoTracker Red (Fig. 4, A and B). The cell-permeant weak base reduced accretion of the dye to background levels.
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Salmonella invasion and intracellular proliferation.
The type of assays developed above can be extended to analyze other forms of particle internalization, such as bacterial invasion into host cells. In contrast to Fc-mediated phagocytosis, where macrophages actively internalize the opsonized particles, during Salmonella invasion the bacterium induces its own entry into the host cell. To analyze this phenomenon in a large population of cells, we implemented a high-throughput method to detect invasion. Cultured HeLa cells were exposed to a strain of Salmonella transformed to express the mRFP1 (Fig. 5B) to enable detection of total bacteria by fluorescence microscopy. As in the case of beads, outside bacteria were visualized after treatment with an anti-Salmonella antibody followed by a Cy2-conjugated secondary antibody (Fig. 5C), while the cells were fixed and identified by nuclear staining (Fig. 5A). Red fluorescent bacteria lacking Cy2, i.e., those that had successfully invaded the cells, were readily detectable (cf. Fig. 5, B and C). Invasion of mammalian cells by Salmonella is independent of PI3K (13), even though, similar to phagocytosis, it is heavily reliant on actin remodeling (7, 11). Knowledge of these properties of the invasive process enabled us to test the validity of the high-throughput assay. Salmonella expressing mRFP1 were incubated for 15 min with HeLa cells that were untreated or pretreated with 100 µM LY-294002 for 30 min or 10 µM CytD for 15 min. The index of invasion, defined as the average number of internalized bacteria per cell, was calculated in several experiments and normalized to the control to simplify comparison. As shown in Fig. 5D, LY-294002 had only a small and statistically insignificant effect on invasion, whereas CytD produced a 91.1% inhibition.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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24,000 individual cells can be imaged and counted under our conditions (Fig. 2F). For comparison, from our experience, it would take a dedicated investigator >30 h of microscope time to manually process the same number of particles. Furthermore, manual counting can introduce systemic error and bias, a problem that is mitigated by use of the automated system. Although the automated imaging capabilities are impressive, the strength of the system lies in its ability to determine diverse physical parameters of the acquired images. First, the software packages can identify and discriminate objects on the basis of user-specified parameters. By differentially labeling associated particles, the software can define and quantify the number of objects with a given label associated with the primary object. In this study, the exclusive labeling of the external beads allowed the SpotDetector BioApplication to rapidly quantify the efficiency of particle internalization (Fig. 1L, Fig. 2, D–F, and Fig. 5D). Information relating to size, shape, and intensity can be concurrently calculated and exported to other statistical software packages. For example, we were able to determine that the average cell size of resting RAW cells labeled with Cell Tracker Green was 520.2 µm2 (Fig. 1B). These morphology measurements can also be used to monitor changes in cell shape and size. Furthermore, more complex target regions can be defined by delineating extended areas with reference to the identified object. In the case of phagosome maturation, the area of a phagosome was established by extending a well-defined, small circular region beyond the perimeter of an internalized bead. Once the region of interest has been defined, fluorescence intensity information from multiple channels can be acquired and processed. In Fig. 4, the acidification of the phagosome was determined using a red acidotropic dye; however, this analysis could be extended to quantify association of any fluorescent molecule with the phagosomes, including fluorescent-protein chimeras. Moreover, information of subcellular localization can be obtained by defining concentric rings of fixed width around an organelle of choice. The displacement of phagosomes, along microtubules, or the intracellular distribution of bacteria can be monitored in this way. We used such a scheme to quantify the subcellular distribution of lysosomes. To this end, using established pulse-chase protocols, we labeled the lysosomes of mouse embryonic fibroblasts with dextran-rhodamine; the cell nuclei were labeled using Hoechst 33342. By measuring the amount of rhodamine fluorescence in multiple concentric rings of fixed width about the nucleus, the distribution of the lysosomes was assessed (see supplemental Fig. S1 in the online version of this article). The distribution of intracellular organelles relative to the plasma membrane could be similarly evaluated by differential labeling of the organellar compartment of interest and the plasma membrane.
Despite its considerable power, the system has some limitations. With the apparatus used here, acquisition is limited to five fluorescent channels with no corresponding bright-field image. The choice of the channel used to acquire the primary objects cannot be modified after acquisition, which may necessitate multiple rounds of image acquisition. Furthermore, because the images are acquired through comparatively thick plastic plates, only objectives with a sufficiently long focal working distance can be used. Only x40 magnification (or less) can be used in this system, which may limit the types of structures that can be optically resolved. Moreover, the ability of the automated microscope to focus on the cells appears to be at times limited by manufacturing defects in the tissue culture plates, which often display uneven thickness. In general, we found that plates with smaller well sizes (e.g., those of a 24-well plate) were more easily handled by the microscope's autofocus algorithms. The autofocusing ability of the microscope also depends on the number of objects in the field under analysis. If objects are too sparse and an empty field is encountered during acquisition, the system will spend considerable time attempting, unsuccessfully, to autofocus. This can significantly increase acquisition time and should be avoided whenever possible. Cells must be plated at a minimum density of 35 and 9 cells/mm2 for imaging at x40 and x20 magnification, respectively, to avoid encountering an empty field. Nevertheless, we found that plating densities 10–40 times the minimum allowed for considerably better focusing and maximized the imaging capabilities of the system.
If an appropriate density of cells is used, along with a small well size, less autofocusing is required, which greatly enhances acquisition speed. On the other hand, overcrowding of particles may present a different problem by interfering with the ability of the software to distinguish individual objects. Even with optimal plating conditions, particles will occasionally be found directly adjacent to each other. In certain software modules, overlapping particles can be discriminated on the basis of geometry or peak intensities. In the former, an algorithm separates touching objects on the basis of parameters of shape, whereas the latter separates objects using the line equidistant from intensity peaks. Although this provides a valuable tool for object discrimination at higher object densities, a suitable number of cells is critical for the experiments to be successful. Experimental conditions must be refined so that the number of particles (e.g., polystyrene beads or bacteria) associated with cells is such that sufficient numbers are present in each field, while difficulties in discriminating between particles due to overcrowding are avoided.
Traditionally, high-throughput techniques have been used to screen large numbers of pharmacological compounds or gene libraries. The automated microscopy system described here should be amenable to such screens, inasmuch as it can be employed to analyze a wide variety of phenotypes in live and fixed cells. Measurements of fluorescence intensity, pattern, and distribution in multiple channels can be applied to study cell division, migration, and survival, as well as low-frequency events, such as transfection of fluorescent chimeras, or to monitor the effectiveness of RNA interference. In this report, we have not only described the use of high-throughput microscopy assays to monitor specific aspects of bacterial invasion and phagocytosis, but we have tried to provide a better understanding of the capabilities of such systems for the study of other cell biological processes. As used here, these assays present only static snapshots of cellular events acquired at defined intervals. However, by virtue of its ability to maintain physiological temperature and atmospheric conditions, together with its robotic capabilities, the KineticScan HCS Reader can, in principle, acquire repeated images of the same cell over time, thus generating detailed kinetic profiles. We foresee that high-throughput microscopy will become a widely used and very powerful research platform for use in basic and translational cell physiology.
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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Present address of C. C. Scott: Dept. of Biochemistry, Sciences II, CH-1211, Geneva, Switzerland.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* These authors contributed equally to this work. 
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REFERENCES |
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