Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

These protocols are designed to understand how human neutrophils interact with and kill bacterial biofilms. The goal is to better understand this, and to limit the variability from assay to assay, understanding that there are inherent issues with human neutrophils from donor to donor variation. The techniques listed here have been optimized to allow the users to perform experiments with minimal variability, especially with loosely attached biofilms.

Additionally, these protocols can also be adapted to study other microbes that can form biofilms. Begin by obtaining isolated colonies of Staphylococcus aureus from a cryopreserved stock using a streak plate technique on a nutrient-rich agar plate, such as tryptic soy agar. Coat individual wells of a 96-well plate with 100 microliters of PLL diluted in sterile water and incubate at room temperature for 30 minutes.

Aspirate the PLL solution aseptically using a vacuum-assisted aspiration trap. Allow the wells to dry overnight at room temperature. Prepare an overnight culture by inoculating a colony of S.aureus in MEM-alpha supplemented with 2%glucose and incubated at 37 degrees Celsius for 16 to 18 hours at 200 rotations per minute.

Dilute the overnight culture by transferring 50 microliters to five milliliters of fresh MEM-alpha supplemented with 2%glucose. Then incubate it at 37 degrees Celsius at 200 rotations per minute until mid-logarithmic phase is achieved. Use MEM-alpha to normalize mid-logarithmic culture to an OD of 0.1.

Transfer 150 microliters of normalized culture to each well of the PLL-treated 96-well plate. Incubate the plate in a humidified chamber at 37 degrees Celsius for 18 to 20 hours. Aspirate the supernatant to remove the planktonic cells.

Gently wash the remaining biomass with 150 microliters of HBSS to remove the unattached cells. Repeat at least twice to remove all the planktonic cells. Add 100 microliters of 20%normal human serum diluted in HBSS dropwise to the washed biofilm and incubate at 37 degree Celsius for 30 minutes to opsonize the biofilm.

Aspirate the serum solution and wash the biofilms dropwise with 150 microliters of HBSS. Aspirate the HBSS, leaving behind wells with opsonized biofilms. Add luminol to the neutrophils re-suspended in HBSS to make up a final concentration of five micromolar luminol.

This solution is ready to use for groups A and C.Add neutrophils mixed with luminol to the wells with opsonized biofilms. For group D, prepare 50 micromolar luminol solution in HBSS in a separate tube without any neutrophils, and add it to the well containing the biofilm. Aliquot 350 microliters of neutrophils mixed with luminol and add PMA at a final concentration of 500 nanograms per milliliter to the mixture.

For group B, add the neutrophils from this mixture into wells without biofilm. This serves as a positive control. Centrifuge the plate at 270 RCF for 30 seconds at four degrees Celsius.

Ensure the plate reader is set to 37 degrees Celsius, the luminescence and kinetic read for 60 minutes with three minute intervals. Place the plate in the plate reader to measure ROS production by neutrophils for 60 minutes. Use a fluorescent strain of S.aureus, such as USA300, expressing GFP to ease microscopy imaging.

Incubate neutrophils with 100 micromolar BCD for 30 minutes in a rocker at 37 degree Celsius with 5%atmospheric carbon dioxide. Ensure the samples are incubated in the dark, and limit light exposure. To wash excess BCD, centrifuge neutrophils at 270 RCF for five minutes, and aspirate the supernatant.

Re-suspend the neutrophils in fresh HBSS. Then add ethidium homodimer-1 to the BCD-stained neutrophils at a final concentration of four micromolar to monitor neutrophil and bacterial death. Wash the biofilm with HBSS and add 150 microliters of neutrophils to the S.aureus biofilm that has been grown in microslides.

Incubate the microslides in a humidified chamber for 30 minutes. The number of bacterial cells will be based on the cell counts obtained from the 18-hour biofilm plating. Image the neutrophil biofilm interaction using fluorescent channels corresponding to fluorescent dyes, or proteins'excitation and emission wavelengths.

Bacterial growth media minimized the viability of neutrophils to approximately 60%after 30 minutes of incubation period. Mammalian cell culture media, however, did not affect the viability of neutrophils, and can also support the growth of S.aureus biofilms. Neutrophils treated with PMA used as a control showed an increased ROS production.

In the absence of biofilms, neutrophils treated with PMA showed robust ROS production. In the presence of S.aureus biofilm, the overall ROS production by neutrophils treated with PMA decreased, while in the absence of PMA, neutrophils solely relied on their interaction with the biofilm, further reducing the ROS production. Widefield fluorescent microscopy revealed that many neutrophils were localized to the surface while a few were within the S.aureus biofilms.

Most of the S.aureus cells interacting with neutrophils were dead, while a few remained alive as determined by LIVE/DEAD staining. Ethidium homodimer-1-stained biofilms revealed a fraction of the dead S.aureus population within the biofilm. The effect of washing the biofilm and neutrophils after 30 minutes of incubation to remove non-adhered neutrophils revealed that around 33%of dead neutrophils were still attached to the biofilm.

The protocols listed here can also be used to further study other functionalities of neutrophils, such as phagocytosis, and formation of neutrophil extracellular traps when neutrophils encounter biofilms.