Commonly, phagocytic assays involve the addition of particles to macrophages followed by microscopic analysis of the number of particles bound and internalized by a cell. This type of analysis is time-consuming as large numbers of cells have to be counted manually to obtain statistically significant results. Therefore, we suggest adapting the assays so that the results may be analysed on a plate reader or flow cytometer which can collect information on large numbers of cells.
There are two types of basic assay. The first determines the number of particles associated with the macrophages, while the other monitors decreasing numbers of particles in the extracellular medium (25). Either assay is acceptable, but we will only discuss the former. Uptake assays can be adapted to measure cellular responses, such as the respiratory burst, by appropriate bulk or single cell methods (26) and to determine the survival or killing of ingested live organisms. Appropriate safety precautions must be taken in handling living micro-organisms, in all procedures.
The ligands expressed on a chosen particle will determine the receptors used for ingestion of that particle and so must be appropriately chosen if investigating a particular receptor. Complex ligands, like bacteria, may be recognized by more than one receptor. Bacteria are easily fluoresceinated using Protocol 9 or some are available commercially (see Table 4). If not investigating phagocytosis mediated by a particular receptor then complex ligands can be used (see Figure 3B). Latex beads, the receptors for which are unknown, are readily taken up and are suitable particles for phagocytosis. They are available in a wide range of sizes and can be coated by absorption or, in the case of carboxylated polystyrene latex beads can be coupled directly to protein ligands to target them to specific receptors; some examples used previously are mannose BSA and lipoarabino-mannan. However, even apparently single ligands may also be recognized by multiple receptors.
Besides latex beads, zymosan, derived from the cell wall of Saccharomyces cerevisiae, is a commonly used particle. It is highly mannosylated and is recognized by a number of receptors including MR, p-glucan receptors, and CR3, with or without opsonization (it readily activates the alternative pathway of complement). It is commercially available, though easy to prepare and label with fluoro-chromes (see Protocol 9). Zymosan should be boiled before use to destroy contaminating phospholipases. Erythrocytes coated with opsonins are widely used to analyse the function of opsonic receptors.
Smaller particles may be taken up by macropinocytosis so when using latex beads ensure that the size used is larger than 1 (Jim in diameter and test the ability of phagocytic inhibitors on particle uptake. Inhibitors of ingestion, like cytochalasin B and D, and inhibitors that block ligand binding should always be used as controls for phagocytosis.
Some ligands require opsonization by complement and antibodies. Macrophages themselves may also produce opsonins like complement and fibronectin that could potentially influence uptake. The presence of serum can opsonize particles, so unless analysing general phagocytosis, use a serum-free protein-containing medium. If analysing specific opsonic receptors, coat the particles with the opsonin before the assay. Bacteria are easily opsonized by incubating them in an appropriate serum for 30 min at 37 °C. Complement is only present in fresh serum and is destroyed by heat inactivation. Specific IgM and complement target CR3, but beware of IgG contamination of the IgM. IgG-coating targets the Fc receptors. Polyclonal Abs can be raised or where available, monoclonal Abs against erythrocyte antigens or hapten, for example, used with an appropriate isotype matched Ab control.
The differentiation of intracellular particles and those bound to the extracellular surface is crucial in any phagocytic assay and there are numerous modifications to existing methods available for this purpose. First, fluorescence on any extracellular particles can be quenched with appropriate agents such as ethidium bromide, crystal violet, and trypan blue. It is not easy to control for total quenching of the extracellular fluorescence. In addition, the quenching agent must not be cell permeable.
When measuring the internalization of live bacteria, antibiotics such as Gentamycin are often added to the culture medium to kill any non-phagocytosed bacteria. Like the quenching agents, the antibiotic should not gain access to the cell where it can kill intracellular bacteria. There is some question, however, as to whether the antibiotic may kill bacteria tightly bound to the plasma membrane or whether endocytically active cells, such as macrophages, also take up the drug.
An alternative approach is to cleave or lyse the bacteria from the extracellular surface, for example, lysostaphin can lyse S. aureus and lysozyme can lyse Micrococcus lysodeikticus. Erythrocytes are easily lysed by brief osmotic shock (water or hypotonic solutions).
Lastly, immunofluorescent techniques can be used to distinguish intra- and extracellular bacteria, with only external bacteria detected by antibodies (see Protocol 11). The distribution of the bacteria, with respect to the numbers found inside and bound to the cell, can be obtained by comparison between antibody staining of permeabilized and unpermeabilized cells.
5.3.5 Time, temperature, and dose
Macrophages are highly professional phagocytic cells and particle ingestion occurs rapidly. Generally, incubation times range between ten minutes and one hour. However, the kinetics of uptake should be determined by performing a time course experiment before embarking on these assays. At time zero there should be no uptake. This would also be true at 4°C as the membrane is not fluid enough to mediate uptake. Following any uptake assay, the macrophages should be quickly cooled to 4°C to stop any further internalization by the macrophage and ideally, the rest of the protocol should be performed in the cold.
The optimal dose of particles should always be determined, especially when using live and virulent bacteria as too many bacteria may lyse or kill the macrophages. An initial particle to macrophage ratio of between 1:1 and 20:1 is recommended. The rate of ingestion should reach zero-order kinetics with increasing dose and is an important test of any assay method.
Particle contact with the macrophages may be enhanced by centrifuging them directly onto the cells, in special holders available commercially. If performing the assay on non-adherent cells, tumble the bacteria and macrophages together for optimal contact. Protocol 11 describes a method of analysis of pahgocytic uptake using adherent populations, though is easily adapted for nonadherent cells.
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