This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold standard for assessing effector functions of antibodies raised against a bacterium as opsonin.
A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.
The opsonophagocytic killing assay (OPKA) is a critical tool for linking alterations in bacterial structure or function to subsequent changes in immune response and function. As such, it is frequently used as a complementary assay to determine immune-based efficacy of antibody treatments, vaccine candidates, enzyme optimization, etc. While in vivo assays are necessary to determine effective clearance or protection in a bacterial infection model, the OPKA can be used to assess immune contribution to bacterial cell death at the most basic components: bacteria, immune cells, and experimental treatments. Previous studies have shown that OPKAs can be modified and used for a variety of bacteria and serotypes, including Streptococcus pneumoniae1, Staphylococcus aureus2, Pseudomonas aeruginosa3. Furthermore, these optimized assays can be used to assess different experimental treatments, including the ability of an enzyme to make the bacterium more accessible to complement-mediated immune cells4 and antibody treatments to improve opsonization5. Classically, OPKA assay has been successfully used in basic and clinical research settings as a powerful indicator for protection induced by pathogen-specific antibodies6,7,8,9.
Different types of immune cells may be used for assessment of opsonophagocytic killing. One commonly used phagocytic population is the HL-60 human leukemic cell line. This cell line can be kept as inactivated promyelocytes in culture; however, they can be differentiated into various activated states via different drug treatments10,11. Treatment of HL60 with N,N-dimethylformamide differentiates the cell line into activated neutrophils with strong phagocytic activity11. While HL-60 cells have been optimized and are frequently used for these phagocytosis assays10, other primary polymorphonuclear leukocytes can be used as the immune arm of the experiment12.
Additionally, these assays can be simplified13 or multiplexed14 to look at multiple antibiotic-resistant strains of the bacteria to be tested. The multiplexed method has been made more feasible through the development of software that can efficiently count bacterial colony forming units (CFUs) per spot on an agar plate15. Here, we describe a streamlined method using one bacterial strain, HL-60 cells, baby rabbit complement, and blood agar plates. With this method, multiple treatments can be assessed quickly to address specific research questions on how the innate immune response to bacterial infection can be modulated.
1. Culture, Differentiation, and Validation of HL-60 Cells
2. Preparation of OPKA Buffers and Reagents
3. Preparation of Bacterial Stock Samples
4. Bacterial Treatment and Culture
5. HL-60 bacterial Co-culture
6. Sample Plating and Overnight Incubation
Validation of HL-60 differentiation should be performed before starting the OPKA. This can be accomplished using flow cytometry to determine the extracellular expression of CD11b, CD35, CD71, and annexin V (Figure 1). Propidium iodide can also be used as a viability marker. After being treated with DMF for 3 days, expression of CD35 should be increased (≥55% of all cells) and expression of CD71 should be decreased (≤20% of all cells). The percentage of annexin V+ and propidium iodide (PI+) cells together should be <35% to ensure sufficient cell viability. If these percentages do not meet the minimum requirements, the culture conditions should be adjusted as described in the Discussion.
The number of CFUs obtained from step 6.5 can be used to compare the bacterial cell survival of different groups compared to the untreated control group (100% cell survival) as shown in Figure 2. For example, the average counts obtained from wells that received no treatment but were co-cultured with HL-60 should be relatively close in number to the cells that received no treatment and no co-culture of HL-60, which would be indicative of 100% cell survival, or 0% cell death. With an effective treatment, the numbers of colonies should be more different between HL-60 co-culture and no HL-60 cells (Figure 2 and Figure 3). Larger differences between HL-60 and no HL-60 sets are indicative of more efficient phagocytosis. However, the treatment may actually improve bacterial cell growth in the set that is not co-cultured with HL-60 cells. This difference in treated and untreated samples should be noted. If the bacterial dilution is not optimized (step 3.6) or the colony growth is not carefully observed after plating (step 6.5 or see Discussion), overgrowth of the colonies may prevent accurate counting of colonies (Figure 4).
Figure 1: Validation of HL-60 cell differentiation via flow cytometry. Differentiated HL-60 cells were harvested, washed, and resuspended in 1 x 105 cells/mL PBS. Cells were then aliquoted into 12 wells (100 µL/well) in a 96-well plate. Cells were then stained with fluorescently conjugated anti-CD35, anti-CD71, annexin V, and propidium iodide. Unstained cells or cells stained with fluorescently conjugated isotype antibodies were used as controls. Please click here to view a larger version of this figure.
Figure 2: Treatment Y improves HL-60-mediated cell killing of bacteria. S. pneumoniae samples were treated with treatment X (antibody) or treatment Y (enzyme). OPKA was performed according to the protocol and bacterial CFUs were counted in duplicate. Samples that were not treated with HL-60 cells were used as a control (100% cell survival). Shown are the average percentages of bacterial CFUs in the HL-60 treated groups compared to the corresponding non-HL-60-treated groups. Bars represent standard error. Please click here to view a larger version of this figure.
Figure 3: Bacterial CFUs after OPKA and overnight culture. Bacterial samples were treated and co-cultured without (A) or with (B) HL-60 cells for 1 h at 37 °C. Samples were diluted according to the protocol and plated on blood agar plates overnight at 30 °C (no CO2). Please click here to view a larger version of this figure.
Figure 4: Bacterial CFU overgrowth. Bacterial samples were treated and OPKA was performed. Samples were diluted and plated on blood agar plates overnight at 37 °C (no CO2). Accurate assessment of colony numbers cannot be determined as overgrowth of colonies is shown. As 37 °C (no CO2) led to bacterial overgrowth, the incubation temperature for future plates was lowered to 30 °C (no CO2) to maintain countability of the colonies. Please click here to view a larger version of this figure.
OPKAs serve essential roles in assessing antibody mediated immune responses induced by vaccinations6,8. The main significance of this simplified OPKA is the adaptability in the conditions to be tested (i.e., antibodies, enzyme treatments, etc.). In this sense, while this assay can be used to test the contribution of opsonins (i.e., antibodies) in phagocytosis, it can also be used to assess ways to overcome virulence factors (i.e., capsular polysaccharides) that normally inhibit phagocytic pathways. Minimizing the number of steps that are typically used in a multiplexed OPKA potentially minimizes the chances for technical errors that can affect the experimental results and reduces the amount of troubleshooting and optimization for obtaining usable data. As this protocol is suited for variations to treatment conditions, it allows for a great deal of versatility.
Pre-establishing the assay through culture of HL-60 and establishment of bacterial stocks is important to prevent extraneous optimization steps when performing the OPKA. Time must be dedicated to making sure all reagents and cell types are ready and functional before the experiment is performed. These steps include propagating the HL-60 cell line in culture (about two weeks), validating that specific concentrations of DMF effectively differentiate the HL-60 cells (about one week), and establishing contaminant-free and optimized bacterial stock dilutions (about two weeks).
Some steps of this protocol are critical for obtaining countable colonies and adequate data. This protocol uses HL-60 cells as phagocytes due to the ease of using a human cell line that can be maintained in culture and differentiated with relatively few steps. Human peripheral blood mononuclear cells (PBMCs) may also be used; however, obtaining these cells and optimizing the conditions for their use may be more challenging. The HL-60 cells must be differentiated in order to function as phagocytes against the bacteria. To verify differentiation after the 3 day treatment with DMF, flow cytometry should be used to test for the expression of CD11b and CD35 on a majority of the cells before any OPKA is attempted, as discussed in step 1.5. Cell viability should also be verified (preferably with annexin V and propidium iodide staining). If a large number of cells are dead, apoptotic, or undifferentiated as observed with flow cytometry, the 3 day differentiation with 0.6% DMF in RPMI media can be modified (0.4%−0.8% DMF, 2−6 day culture time) until cell viability and differentiation markers are improved. This differentiation should be the first optimization of the OPKA as HL-60 function is critical for effective bacterial killing. We recommend validating HL-60 differentiation before every OPKA experiment.
The number of bacterial CFUs (step 3.6) initially dispensed into the 96-well plate (step 4.2) is also critical: dispensing too many cells will make counting difficult and inaccurate (step 6.5) and may decrease the cell death observed from HL-60 co-culture, whereas dispensing too few cells may increase the amount of deviation between duplicates and may not show any countable colonies after HL-60 co-culture. Optimizing the stock dilution is therefore critical, and must be tested with the full protocol, including HL-60/complement co-culture.
The importance of complement may also be tested with this protocol by including two sets of samples: one with active baby rabbit complement and one with heat-inactivated complement. HL-60 cells should be co-cultured with both sets, though a bacteria-only set should still be included as a 100% cell survival baseline.
For some bacterial serotypes, the morphology of the colonies may make cell counting or visibility problematic. Mucoid serotypes such as type 3 Streptococcus pneumoniae, for instance, can easily overgrow and reduce the CFU counts. This may prove especially problematic when testing treatments that affect the capsule, as overgrowth would be prevented in the treated group but greater number of smaller colonies would be counted. To prevent this discrepancy, control of cell growth is critical. Culturing the plated colonies at 30 °C overnight will likely allow for improved monitoring of bacterial growth and the plates can be removed when all colonies reach distinguishable, countable sizes. Additionally, different agar plates may be used to improve visibility of individual colonies. In this way, this protocol is advantageous as small changes to optimize conditions can thus be used to account for a number of bacterial strains or various treatment options.
The authors have nothing to disclose.
We thank Dr. Moon Nahm (University of Alabama Birmingham) for his invaluable assistance in establishing OPKA assays in our laboratory. This work was supported by National Institutes of Health Grant 1R01AI123383-01A1 to FYA.
Annexin V (APC conjugated) | BioLegend | 640919 | |
anti-CD35, human (PE conjugated) | BioLegend | 333405 | |
anti-CD71, human (PE conjugated) | BioLegend | 334105 | |
bacterial strain to be used (ie, Streptococcus pneumoniae, WU2) | Bacterial Respiratory Reference Laboratory (Dr. Moon Nahm) | ||
blood agar plates | Hardy Diagnostic | A10 | |
Fetal Clone serum | HyClone | SH30080.03 | |
glycerol | Sigma | G9012-1L | |
HL-60 cells | ATCC | CCL-240 | |
IgG Isotype Control (PE conjugated) | BioLegend | 400907 | |
N,N-dimethylformamide (DMF) | Fisher Chemical | UN2265 | |
propidium iodide | Sigma | P4864 | |
RPMI media with L-glutamine | Corning | 10-040-CV |