Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

This assay links modulation of bacterial structure and function to immune cell based antibacterial activity. As such, a key immunotherapeutic of a potential treatment can be assessed in determining the likelihood of improved bacterial clearance. Opsonophagocytic killing assay traditionally serves as the gold standard for assessing effective functions of antibodies raised against the bacterium as obsonant.

This protocol offers simplicity and versatility over existing high throughput standardized assays. In this protocol, we investigate the effects of a drug treatment on streptococcus pneumonia and how these effects translate to improved phagocytosis of the bacterium. Here, we apply the OPKA to streptococcus pneumonia cells.

However this protocol can be adopted to assess phagocytic killing of other pathogens by immune cells. Dedicate time to optimize the bacterial stocks and ensure the final CFUs to be counted fall within the optimum range. We strongly recommend using flow cytometry to ensure HL60 cells are viable and active and trying different culture conditions to ensure functionally active cells.

Visual demonstration will give a better understanding of how the sample should be plated and how to achieve countable samples. To begin, prepare HL60 cell culture media composed of 500 milliliters RPMI supplemented with L-glutamine and 15 milliliters heat inactivated fetal bovine serum. For propagation and maintenance of HL60 cells, culture five million cells in 10 milliliters of HL60 cell culture media in 75 square centimeter vented flasks at 37 degrees celsius and five percent carbon dioxide.

To generate working stocks of HL60 cells, add approximately one million cells per milliliter in HL60 culture media supplemented with 10%dimethyl sulfoxide into one milliliter cryogenic tubes. To differentiate HL60 cells, culture 1.5 times 10 to the seven cells in 15 milliliters of HL60 cell culture media supplemented with 0.6%DMF in sterile filter capped 75 square centimeter flasks at 37 degrees celsius and 5%carbon dioxide for three days prior to OPKA. To prepare the bacterial stock samples, grow the WU2 bacterial strain in an appropriate broth for approximately two to four hours at 37 degrees celsius.

Next, pellet the bacteria by centrifugation at six thousand times G for two minutes and resuspend the cells in 10 to 30 milliliters of 15%glycerol in the appropriate broth. Aliquot the bacteria culture into sterile 1.5 milliliter centrifuge tubes and store at 80 degrees celsius. Next, thaw out one vial of bacterial stock in a 37 degree celsius water bath.

Pellet the bacterial cells and resuspend in 500 microliters of OBB under sterile conditions. Plate dilutions of the untreated bacterial stock on blood agar plates and culture the plates overnight at 30 degrees celsius. After the incubation step, count the colonies for each dilution of untreated bacterial stock co-cultured with HL60 cells.

Record which dilution of bacteria yields the optimal number of countable colonies for future OPKAs with this bacterial stock. Thaw one tube of bacterial stock. Pellet bacteria at six thousand times G for two minutes and resuspend the cell pellet in OBB at the optimal dilution obtained in the previous step.

Pipette 10 microliters of the resuspended bacterial dilution per well in a round bottom 96 well cell culture plate. Then, add 20 microliters of an appropriate antibody or a drug treatment to each experimental well in duplicate. For control wells, use 1XPBS or OBB depending on the buffer used for the treatment wells.

Shake the sample plate at approximately 90 rpm at room temperature for one hour. To harvest the HL60 differentiated cells that are treated with DMF three days prior, pellet the cells at 500 times G for three minutes. Discard the supernatant and wash the pellet with at least 10 milliliters of 1XPBS.

Pellet the washed cells at 500 times G for three minutes. Discard the supernatant and resuspend the cells in OBB. Add sterile undiluted baby rabbit serum at a one to five final volume.

After a one hour bacterial culture, divide each sample into duplicate wells for two groups. Next, add 50 microliters of the HL60 complement mixture to each experimental set of wells and 50 microliters of OBB to the wells of bacteria only. Shake the 96 well plate at 37 degrees celsius for one hour.

To plate samples, dilute each well with OBB at a one to five final volume so that each sample has a volume of at least 50 microliters. Next, pipette 50 microliters of each sample directly onto a designated area of a bacterial culture plate, ensuring adequate spacing between samples. Cover and allow samples to dry for approximately 15 minutes at room temperature.

Invert the plates and culture overnight at 30 degrees celsius. Alternatively, culture plates in anaerobic jars to test whether anoxic conditions affect the bacterial growth or to control for morphology. After overnight culture, count the colonies in each designated sample area and proceed to analyzing data by comparing the number of live cells in each set to the corresponding controls.

The most difficult aspects of establishing this technique for the first time will likely be optimizing the starting CFUs of the bacterial stock, as well as ensuring the HL60 cells are effectively differentiated. Before starting the OPKA, HL60 differentiation was validated using flow cytometry with propidium iodide as a viability marker. After being treated with DMF for three days, expression of CD35 was increased and expression of CD71 was decreased.

The average percentages of bacterial CFUs in the HL60 treated groups compared to the corresponding non-HL60 treated groups was used to compare the bacterial cell survival of different treatments. Results showed that with an effective treatment, the numbers of colonies were different between HL60 co-culture and no HL60 cells, indicative of more efficient phagocytosis. Results showed that when the bacterial dilution was not optimized or the colony growth was not carefully observed after plating, overgrowth of the colonies could prevent accurate counting of colonies.

The most important thing to remember when attempting this procedure is to monitor the bacterial incubation carefully so as to avoid overgrowth of the CFUs. In vivo assays can be used to asses effective bacterial clearance within a host. These assays can be used to confirm the immune efficacy observed with the OPKA are translatable to human or animal models.

The opsonophagocytic killing assay has been used to great effect in previous significant research studies to link antibacterial therapies to phagostic immune responses. Working with bacterial pathogens can be hazardous. Please wear protective personal equipment at all times during this assay.