An In Vitro Assay to Study the Interaction between Neutrophils and Biofilm

1. Preparation of in vitro biofilm

1. Obtain isolated colonies of S. aureus from a cryopreserved stock using a streak-plate technique on a nutrient-rich agar plate, such as Tryptic Soy Agar (see Table of Materials).
2. Coat individual wells of a 96-well plate with 100 µL of 0.001% (v/v) poly-L-Lysine (PLL) diluted in sterile H₂O and incubate at room temperature for 30 min. Aseptically, aspirate the PLL solution using a vacuum-assisted aspiration trap. Allow the wells to dry overnight at room temperature.
   NOTE: All aspiration steps in the protocol are performed using a vacuum-assisted aspiration trap unless otherwise stated.
3. Prepare an overnight culture by inoculating a colony of S. aureus in minimal essential media alpha (MEMα) supplemented with 2% glucose and incubate at 37 °C, shaking at 200 rpm for 16-18 h.
4. Dilute the overnight culture by transferring 50 µL to 5 mL of fresh MEMα supplemented with 2% glucose and incubate at 37 °C, shaking at 200 rpm, until mid-logarithmic phase, generally between optical density 600 (OD₆₀₀nm) of 0.5-0.8. Use MEMα to normalize mid-logarithmic culture to an OD₆₀₀nm of 0.1.
5. Transfer 150 µL of normalized culture to each well of the PLL-treated 96-well plate. Incubate statically for 18-20 h in a humidified chamber at 37 °C.
   NOTE: The biofilms can also be grown in other formats such as µ-channel slides (see Table of Materials).
6. Aspirate the supernatant to remove the planktonic cells. Gently wash the remaining biomass with 150 µL of Hanks' Balanced Salt Solution (HBSS) to remove the unattached cells. Add HBSS dropwise to avoid disrupting the biofilm.
   NOTE: While aspirating the supernatant and HBSS during washes, leave just enough liquid (supernatant or HBSS) in the wells containing biofilm such that the biofilm is still immersed. This prevents the disruption of the biofilm structure when HBSS is added dropwise to wash the biofilm.
7. Repeat step 1.6 at least two more times to remove all the planktonic cells. At this point, biofilms are ready for immediate downstream experiments.
   NOTE: If the biofilms are not used for neutrophil experiments, HBSS can be substituted with phosphate-buffered saline (PBS). HBSS is preferred over PBS as HBSS contains components, including glucose, that provide optimum conditions for neutrophil activation.

2. Neutrophil isolation

NOTE: Neutrophils were isolated following a previously published method with minor changes. This isolation protocol combines density gradient centrifugation first, followed by 3% dextran sedimentation. This section only covers the overall neutrophil isolation protocol, focusing on the changes made to the published protocol. Furthermore, the protocol outlined below is one of the many methods that can isolate neutrophils and can be substituted as needed. Other methods for isolating neutrophils include the use of cell separation media or magnetic antibody cell separation.

1. Draw blood from an adult donor via venipuncture, as per the protocol outlined in the institutional IRB. Prior to the blood draw, ensure that the syringe has sufficient preservative-free heparin, such that the final concentration of heparin is 20 U/mL.
2. Dilute the heparinized blood with 3/4 the volume of endotoxin-free 0.9% NaCl (see Table of Materials) in H₂O at room temperature.
3. For every 20 mL of the diluted blood sample, aliquot 14 mL of a commercially available density gradient medium (see Table of Materials) in a fresh 50 mL conical tube. Carefully layer the diluted blood sample on top of the density gradient medium.
4. Centrifuge the layered blood sample at 400 x g for 40 min at room temperature. Ensure that the centrifuge has a slow break to avoid disturbing the layer once the centrifugation is completed.
   NOTE: The blood sample will have five layers containing a mixture of saline and plasma, a mononuclear cell layer, a density gradient medium, neutrophils, and erythrocytes.
5. Using a serological pipette, aspirate all the layers above the neutrophils and erythrocyte pellet, followed by a gentle resuspension of the pellet in cold endotoxin-free 0.9% NaCl in H₂O. For each pellet generated from a 20 mL blood sample, resuspend the pellet back to 20 mL volume total. Add 1:1 volume of 3% dextran (see Table of Materials). Incubate the tube upright for 18-20 min on ice.
   NOTE: Ensure that the 3% dextran is made with endotoxin-free 0.9% NaCl in H₂O.
6. Remove 20 mL of the upper layer that contains neutrophils and some erythrocytes onto a new 50 mL conical tube and centrifuge it at 355 x g for 10 min at 4 °C. Pour off the supernatant leaving behind a red pellet.
7. Gently resuspend the pellet in 10 mL of cold, sterile H₂O for 30 seconds to lyse the remaining erythrocytes. Immediately add 10 mL of cold endotoxin-free 0.9% saline to the mixture to restore tonicity. Centrifuge the solution at 233 x g for 3 min at 4 °C.
8. Pour off the supernatant and resuspend the pellet containing 95%-97% neutrophils in 1 mL cold HBSS per 20 mL of the blood sample.
9. Transfer 10 µL of the resuspended neutrophils in 90 µL of 0.4% trypan blue exclusion dye and count the cells using a hemocytometer (see Table of Materials).
   NOTE: Non-viable cells are stained blue as trypan blue exclusion dye is impermeable in viable cells. This protocol provides >99% cell viability.
10. Add additional HBSS such that the final concentration of neutrophils is 4 x 10⁶ cells/mL.
    NOTE: For instances with <99% cell viability, the final concentration of 4 x 10⁶ cells/mL can still be achieved; however, the total volume of solution containing 4 x 10⁶ cells/mL obtained will decrease. The final concentration of neutrophils can be adjusted according to the user's experimental needs. The neutrophils were resuspended at a 4 x 10⁶ cells/mL final concentration for all the experiments described below. To account for donor-to-donor variability, it is strongly recommended that all experiments involved with neutrophils be performed with at least three different donors.

3. Imaging biofilm-neutrophil interactions

1. Set up a biofilm using steps 1.2-1.6. To facilitate biofilm imaging, employ a fluorescent strain of S. aureus, such as USA300 expressing green fluorescent protein (GFP), to increase the ease of microscopy imaging.
   NOTE: A 6 µ-channel slide (see Table of Materials) was used instead of a 96-well plate to demonstrate the in vitro biofilm model (step 1).
2. Incubate 4 x 10⁶ cells/mL of neutrophils with 100 µM of Blue CMAC (7-amino-4-chloromethylcoumarin) Dye (BCD, see Table of Materials) for 30 min in a rocker at 37 °C and 5% CO₂. Ensure the samples are incubated in the dark and limit exposure to light for the remaining steps.
3. To wash excess BCD, centrifuge neutrophils at 270 x g for 5 min and aspirate the supernatant. Resuspend the neutrophils in fresh HBSS. At this point, add ethidium homodimer-1 (see Table of Materials) to the BCD-stained neutrophils at a final concentration of 4 µM to monitor neutrophil and bacterial death.
4. Add 150 µL of neutrophils to the S. aureus biofilm that has been grown in µ-slides, such that the neutrophil to bacteria ratio is 1:30 (neutrophil: bacteria). Incubate the µ-slides in a humidified chamber for 30 min. The number of bacterial cells is based on the cell counts obtained from plating an 18 h biofilm.
5. Image the neutrophil-biofilm interaction using fluorescent channels corresponding to the excitation and emission wavelengths of the fluorescent dyes/proteins.
   NOTE: For the present study, BCD is 353/466 nm, ethidium homodimer-1 is 528/617 nm, and GFP is 395/509 nm. Limit the exposure of the sample to the laser or the light to prevent photobleaching of the samples.
6. Analyze the images using microscopy image analysis software or programs such as FIJI/ImageJ, COMSTAT2, BiofilmQ, and BAIT, among many more.
   NOTE: When working with stains, it is important to consider the specificity of the dyes in use. Some stains work on prokaryotic and eukaryotic cells, while others work only on one. If neutrophils and biofilms are separately stained using dyes that can stain both cell types, ensure to wash off any remaining dye before combining neutrophils and biofilms to prevent cross-staining.