This work was funded by the National Institute of Allergy and Infectious Diseases (R01AI077628) to DJW and an American Heart Association Career Development Award (19CDA34630005) to ESG. We thank Dr. Paul Stoodley for providing us with USA 300 LAC GFP strain. Furthermore, we acknowledge resources from the Campus Microscopy and Imaging Facility (CMIF) and the OSU Comprehensive Cancer Center (OSUCCC) Microscopy Shared Resource (MSR), The Ohio State University. We also thank Amelia Staats, Peter Burback, and Lisa Coleman from the Stoodley lab for performing blood draws.

All procedures were approved by the Ohio State University Institutional Review Board (IRB) (2014H0154). Informed written consent was obtained from all the donors for collecting peripheral blood to isolate primary human neutrophils. Staphylococcus aureus (USA300 LAC)31 was used as the model organism for performing the experiments. The experiments were performed with proper personal protective equipment (PPE) due to potential exposure to a bloodborne pathogen.
1. Preparation of in vitro biofilm
<ol>
	<li>Obtain isolated colonies of S. aureus from a cryopreserved stock31 using a streak-plate technique32,33 on a nutrient-rich agar plate, such as Tryptic Soy Agar (see Table of Materials).</li>
	<li>Coat individual wells of a 96-well plate with 100 &#181;L 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.</li>
	<li>Prepare an overnight culture by inoculating a colony of S. aureus in minimal essential media alpha (MEM&#945;) supplemented with 2% glucose and incubate at 37 &#176;C, shaking at 200 rpm for 16-18 h.</li>
	<li>Dilute the overnight culture by transferring 50 &#181;L to 5 mL of fresh MEM&#945; supplemented with 2% glucose and incubate at 37 &#176;C, shaking at 200 rpm, until mid-logarithmic phase, generally between optical density 600 (OD600nm) of 0.5-0.8. Use MEM&#945; to normalize mid-logarithmic culture to an OD600nm of 0.1.</li>
	<li>Transfer 150 &#181;L 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 &#176;C. 
	NOTE: The biofilms can also be grown in other formats such as &#181;-channel slides (see Table of Materials).</li>
	<li>Aspirate the supernatant to remove the planktonic cells. Gently wash remaining biomass with 150 &#181;L of Hanks&#39; 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.</li>
	<li>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. 
	&#8203;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 activation34.</li>
</ol>
2. Quantification of biofilm biomass
<ol>
	<li>Prepare a stock of 0.1% (w/v) Crystal Violet (CV) solution (see Table of Materials) by dissolving in 20% (v/v) ethanol and 80% (v/v) H2O. Ensure that the CV is completely dissolved in ethanol prior to adding H2O. Filter-sterilize the solution.</li>
	<li>Add 150 &#181;L of 0.1% CV solution to the washed biofilm and incubate for 20 min at room temperature. Use at least three empty wells as media-only controls.</li>
	<li>Aspirate the 0.1% CV solution from the biofilms and wash the stained biofilms with 200 &#181;L of 1x PBS. Repeat this process for a total of three washes to remove any excess CV from the wells.</li>
	<li>Add 150 &#181;L of 33% (v/v) glacial acetic acid diluted with H2O. Incubate at room temperature on a rocker at 50 rpm for 30 min to allow the CV bound to the biomass to dissolve completely. 
	CAUTION: Perform this step in a laminar flow hood with appropriate PPE as glacial acetic acid is a corrosive chemical.</li>
	<li>In the meantime, set up the microplate reader (see Table of Materials) to read the CV stain values. Following glacial acetic acid treatment, read the plate at 595 nm wavelength. 
	&#8203;NOTE: The wavelength used to measure the OD of CV can range from 500-600 nm35.</li>
</ol>
3. Neutrophil isolation
NOTE: Neutrophils were isolated following a previously published method with minor changes36. 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 separation37.
<ol>
	<li>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.</li>
	<li>Dilute the heparinized blood with 3/4 the volume of endotoxin-free 0.9% NaCl (see Table of Materials) in H2O at room temperature.</li>
	<li>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.</li>
	<li>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, density gradient medium, neutrophils, and erythrocytes.</li>
	<li>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.</li>
	<li>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 &#176;C. Pour off the supernatant leaving behind a red pellet.</li>
	<li>Gently resuspend the pellet in 10 mL of cold, sterile H2O for 30 s 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 &#176;C.</li>
	<li>Pour off the supernatant and resuspend the pellet containing 95%-97% neutrophils in 1 mL cold HBSS per 20 mL of the blood sample.</li>
	<li>Transfer 10 &#181;L of the resuspended neutrophils in 90 &#181;L 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 &#62;99% cell viability37,38.</li>
	<li>Add additional HBSS such that the final concentration of neutrophils is 4 x 106 cells/mL. 
	NOTE: For instances with &#60;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&#39;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.</li>
</ol>
4. Measurement of ROS produced by neutrophils
<ol>
	<li>Add 100 &#181;L of 20% normal human serum (diluted in HBSS) dropwise to the washed biofilm (step 1.6) and incubate at 37 &#176;C under static condition for 30 min to opsonize the biofilm.</li>
	<li>Aspirate the 20% serum solution and wash the biofilms dropwise with 150 &#181;L of HBSS once. Aspirate the HBSS, leaving behind wells with opsonized biofilms. 
	NOTE: For interpretation of the experiment, a minimum of four groups is recommended: (A) Neutrophils + Biofilm, (B) Neutrophils + PMA (positive control, see Table of Materials), (C) Neutrophils only, and (D) Biofilm only.</li>
	<li>Add luminol (see Table of Materials) to the neutrophils resuspended in HBSS at a concentration of 4 x 106 cells/mL such that the final luminol concentration is 50 &#181;M. This solution is ready to use for groups (A) and (C). Add 4 x 105 neutrophils mixed with luminol to the wells with opsonized biofilms.</li>
	<li>In a separate tube, prepare 50 &#181;M luminol solution in HBSS without any neutrophils and add it to the well containing biofilm (group D).</li>
	<li>Aliquot 350 &#181;L of neutrophils mixed with luminol and add phorbol 12-myristate 13-acetate (PMA) at a final concentration of 500 ng/mL to the mixture. For group (B), add 4 x 105 neutrophils from this mixture into wells without biofilm. This serves as a positive control. 
	NOTE: The concentration of PMA indicated in this step is relatively high to ensure robust burst response as PMA stimulated neutrophils is a positive control. PMA can be used at a lower concentration to activate neutrophils, depending on the experiment.</li>
	<li>Centrifuge the plate at 270 x g for 30 s at 4 &#176;C.</li>
	<li>Ensure the plate reader is set to 37 &#176;C along with the setting for luminescence and kinetic read for 60 min with 3 min intervals. Place the plate in the plate reader to measure ROS production by neutrophils for 60 min. 
	&#8203;NOTE: For this assay, biofilms were grown in white plates used for luminescence assays. PMA is a known agonist for the oxidative burst response39. When performing studies involving PMA, ensure that PMA is added at the final step while the solution containing neutrophils is cold since PMA immediately initiates the burst response.</li>
</ol>
5. Imaging biofilm-neutrophil interactions
<ol>
	<li>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)40,41, to increase the ease of microscopy imaging. 
	NOTE: A 6 &#181;-channel slide (see Table of Materials) was used instead of a 96-well plate to demonstrate the in vitro biofilm model (step 1).</li>
	<li>Incubate 4 x 106 cells/mL of neutrophils with 100 &#181;M of Blue CMAC (7-amino-4-chloromethylcoumarin) Dye (BCD, see Table of Materials) for 30 min in a rocker at 37 &#176;C and 5% CO2. Ensure the samples are incubated in the dark and limit exposure to light for the remaining steps.</li>
	<li>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 &#181;M to monitor neutrophil and bacterial death.</li>
	<li>Add 150 &#181;L of neutrophils to the S. aureus biofilm that has been grown in &#181;-slides, such that the neutrophil to bacteria ratio is 1:30 (neutrophil: bacteria). Incubate the &#181;-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.</li>
	<li>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.</li>
	<li>Analyze the images using microscopy image analysis software or programs such as FIJI/ImageJ, COMSTAT2, BiofilmQ, and BAIT, among many more42,43,44,45. 
	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.</li>
</ol>

The authors have nothing to disclose.

There have been numerous efforts to grow robust and reproducible S. aureus biofilms for downstream experiments in vitro48,49,50. A standardized protocol is outlined that takes advantage of the cationic nature of PLL, as well as supplementing the media with glucose for the growth of robust in vitro S. aureus biofilms. The addition of PLL allows for better attachment of the negatively charged bacterial cell to the positively charged PLL coated surfaces. It is important to note that PLL at a 10 &#181;g/mL concentration has antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, and S. aureus when incubated for 24 h51. The same concentration is used to coat surfaces; however, excess PLL is aspirated, making the concentration of PLL lower than 10 &#181;g/mL when seeding for biofilm growth.
It is important to note that PLL has worked only in specific growth media such as MEM&#945; with 2% glucose, where it was observed that S. aureus produced robust biofilms with minimal variability (Figure 2A). PLL concentration to be used in conjunction with other media types would require further optimization, such as using an increased concentration of PLL to coat the wells. Additionally, these conditions have been optimized for a monospecies S. aureus biofilm. While chronic wound biofilms are often polymicrobial, standardizing assays to study monospecies biofilm and its interactions with neutrophils and other immune cells is key in understanding their contribution to pathogenesis52. These standardized protocols can be optimized further to sustain and study polymicrobial biofilms and their interactions with neutrophils.
It was also observed that rich bacterial culture media, such as TSB, led to a loss of neutrophil viability (Figure 1). Therefore, growth conditions of S. aureus biofilms in MEM&#945;, used for mammalian cell cultures, were optimized. For studies involving neutrophils, this media supports neutrophil viability and promotes S. aureus growth. While it was observed that media affects the viability of neutrophils, it is also important to consider that neutrophils isolated from peripheral human blood undergo apoptosis ex vivo with approximately 70% apoptotic neutrophils by 20 h53. This necessitates proper handling, such as storing the neutrophils on ice when preparing for experiments, using endotoxin-free reagents, and preventing activation of neutrophils by avoiding vortexing of samples with neutrophils.
The assessment of oxidative burst in neutrophils is routinely performed to determine the killing effect of neutrophils on the pathogen14,54,55. These studies are frequently performed with planktonic bacteria where neutrophils are added, and the oxidative burst response is quantified using luminol-amplified chemiluminescence that detects superoxide anions produced by neutrophils. The present protocol is modified by replacing planktonic bacteria with statically grown 18 h S. aureus biofilm. As such, neutrophils can be directly added to the biofilm to assess their activation. On the other hand, bacteria in biofilms produce enzymes, such as catalase and superoxide dismutase to detoxify ROS23,56. Staphylococcus epidermidis biofilms produce higher catalase than its planktonic counterpart under stress57. The total chemiluminescence of PMA-stimulated neutrophils in an S. aureus biofilm is significantly lower than the PMA-stimulated neutrophils where biofilm is absent (Figure 2). This may be due to the activity of these detoxifying enzymes. Furthermore, S. aureus biofilms produce several pore-forming toxins called leukocidins that kill neutrophils58. The reduced burst response is also likely due to the reduced viability of neutrophils in the presence of S. aureus biofilm. While this study uses luminol that detects the total ROS produced both inside and outside of the cells, other reagents, such as CM-H2DCFDA (5-(and-6)-chloromethyl-2&#39;7&#39;-dichlorodihydrofluorescein diacetate) or isoluminol, need to be considered if the goal of the work is to study intracellular or extracellular ROS production14,53,54 specifically.
The ability to visualize neutrophil-biofilm interactions via microscopy can be informative about the behavior of neutrophils and biofilms in the presence of each other. The excitation and emission spectra of the fluorescent dyes and proteins represent a snapshot of the interaction between an 18 h S. aureus biofilm and neutrophils after a 30 min incubation. To effectively capture signals from stained cells, it is important to limit exposure of the samples to light sources while setting up the samples for microscopy. While imaging, rapid photobleaching of the samples was avoided by lowering the intensity of the light source when adjusting all the parameters such as Z-stack height and exposure time for different channels.
These simple practices allowed for proper microscopy imaging where it was observed that few neutrophils are localized within the biofilm (Figure 4A). This may be due to spaces present within the biofilm as 18 h S. aureus biofilm grown in MEM&#945; with 2% glucose does not uniformly cover the surface (Figure 4B). However, other studies&#39; use of rich media has shown a uniform lawn of S. aureus biofilm growth and leukocytes penetrating through the&#160;biofilm30,58. Furthermore, it is also observed that there was neutrophil cell death after 30 min of incubation with S. aureus biofilms due to S. aureus biofilm-produced leukocidins that lyse neutrophils58 (Figure 4A,D). Addition of a wash step to remove non-adhered neutrophils after incubating them with biofilm for 30 min removed ~15% of dead neutrophils from the system compared to the unwashed group, in which microscopy was performed immediately after 30 min of incubation (Figure 4D). Neutrophils interacting with S. aureus were also observed (Figure 4C). Further experiments are required to assess whether S. aureus is engulfed by neutrophils or attached to the cell surface of neutrophils54.&#160;Imaging neutrophils and biofilms is the first step&#160;to evaluate several neutrophil functionalities downstream, such as phagocytosis and NETosis54,59. The effect of neutrophils on biofilms can also be assessed by quantifying the biofilm biomass, structural changes of the biofilm, and biofilm viability, among many others, using image analysis tools listed in step 5.6. Lastly, donor-to-donor variability exists in neutrophils; thus, it is recommended that at least three different donors be used for studies involving neutrophils.
Overall, standardized in vitro assays were combined to assess interactions between neutrophils and biofilms. Though these assays utilize S. aureus, the protocols described can easily be adapted to study other pathogens. While there are various in vivo models to study host-pathogen interactions, they can be expensive and labor-intensive, especially if the conditions are not optimized. Working with standardized in vitro assays allows one to optimize experimental conditions and confirm observations prior to moving to an in vivo system. Finally, various animal infection models have been used to study biofilm-neutrophil interactions in vivo. However, it is important to consider immunological differences between humans and animal models60,61,62,63. This necessitates using neutrophils derived from humans to study these complex host-pathogen interactions.

A biofilm is a community of surface-associated microbes or non-attached aggregates encased in an extracellular polymeric substance (EPS)1,2. These communities protect the encased microorganisms from environmental stressors, including tolerance to antimicrobial agents and the immune system3. Several pathogenic microbial species form biofilms that have been associated with chronic infections4. The development of biofilms is an intricate process involving attachment to surfaces, EPS production, cell proliferation, biofilm structuring, and cell detachment5. Once cells disperse to form a biofilm, they remain planktonic or translocate to a new substratum and re-initiate biofilm development6.
Staphylococcus aureus, an opportunistic pathogen, follows a general scheme of biofilm development, including attachment, proliferation, maturation, and dispersal7. The attachment process in S. aureus biofilms is dictated by hydrophobic interactions, teichoic acids, and microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)8,9. As the proliferation of S. aureus begins, EPS, which primarily consists of polysaccharides, proteins, extracellular DNA, and teichoic acids, is produced5. As EPS components are produced, various exoenzymes and small molecules are also produced, contributing to the biofilm 3-dimensional structure and aiding in detachment5. S. aureus takes advantage of this highly coordinated lifestyle to establish various chronic infections, including infections due to the indwelling of medical devices10.
Methicillin-resistant S. aureus (MRSA) is one of the leading causes of infections related to indwelling medical devices, such as central venous and urinary catheters, prosthetic joints, pacemakers, mechanical heart valves, and intrauterine devices11. During such infections, neutrophils are the first host immune cells recruited to the infection site to combat pathogens via multiple strategies12. These include phagocytosis, degranulation, reactive oxygen and nitrogen species (ROS/RNS) production, or release of neutrophil extracellular traps (NETs) to eliminate pathogens13.
Generation of ROS upon phagocytosis of microbes is one of the key antimicrobial responses exhibited by neutrophils14. Phagocytosis is enhanced if microbes are coated in opsonins, particularly immunoglobulins and complement components found in serum15. The opsonized microbes are then recognized by cell surface receptors on neutrophils and engulfed, forming a compartment called the phagosome15. Neutrophils generate and release ROS in the phagosome via the membrane-associated NADPH-oxidase16. This multi-component enzyme complex generates superoxide anions by transferring electrons to molecular oxygen16. Additionally, neutrophils also generate RNS through the expression of inducible nitric oxide synthase (iNOS)17. These high superoxide and nitric oxide radicals within the phagosome have broad antimicrobial activities. They can interact with metal centers in enzymes and damage nucleic acids, proteins, and cell membranes of the pathogen18,19,20,21. Numerous microbes adopt a biofilm lifestyle and employ different strategies to evade killing by ROS22,23. Thus, standardized assays that couple biofilms with neutrophils to quantify ROS are beneficial for consistent results.
While assays, such as quantifying neutrophil ROS production, provide information&#160;about the responses of neutrophils to biofilms, the ability to visualize the interactions of neutrophils within a biofilm can also serve as a powerful tool. The use of fluorescent dyes for microscopy often requires optimization to obtain high-quality images that can be used for microscopy imaging analysis. The flexibility to optimize some conditions is limited as neutrophils can undergo cell death post-isolation. Furthermore, biofilms are typically washed to remove the planktonic population from the experimental set-up before the addition of neutrophils. While washing, variability between replicate biofilms may arise due to loss of partial biomass if biofilms are loosely adhered to the surface.
Broadly, current methods in the field to analyze interactions between neutrophils and biofilms mainly include microscopy, flow cytometry, and colony-forming units (CFU) enumeration24,25,26,27. Microscopy involves the use of dyes that either directly stain the neutrophils and biofilms, or target various neutrophil responses against microbes such as NET formation, degranulation, and cell death25,28. A subset of these responses, such as neutrophil cell death and degranulation, can also be analyzed via flow cytometry, but requires neutrophils to be preferentially unassociated with large aggregates of microbes in a biofilm28,29. Flow cytometry can also quantify some biofilm parameters, such as cell viability27. These processes, however, require disruption of the biofilm biomass and would not be useful to visualize other important interactions such as the spatial distribution of neutrophils and their components within a biofilm27,29,30.
The present protocol focuses on adapting some of the traditionally used methods to study neutrophil-biofilm interactions on biofilms that have been optimized to provide minimal variability during handling. This protocol thus provides standardized methods to grow and quantify biofilms, isolate primary human neutrophils from peripheral blood, quantify ROS production, and visualize biofilm-neutrophil interactions via microscopy. This protocol can be adapted to different systems to understand biofilm-neutrophil interactions while considering the heterogeneity among donor pools.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% sodium chloride irrigation, USP</td>
			<td>Baxter</td>
			<td>2F7124</td>
			<td>Endotoxin-free; Used for isolation of neutrophils</td>
		</tr>
		<tr>
			<td>150 mL rapid-flow filter unit</td>
			<td>Thermo Scientific</td>
			<td>565-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>200 proof ethanol</td>
			<td>VWR</td>
			<td>89125-188</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>BD</td>
			<td>309657</td>
			<td>Used for blood draw</td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tubes</td>
			<td>Thermo Scientific</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mL syringe</td>
			<td>BD</td>
			<td>309653</td>
			<td>Used for blood draw</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher Bioreagents</td>
			<td>BP1423-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol swab</td>
			<td>BD</td>
			<td></td>
			<td>Used for blood draw</td>
		</tr>
		<tr>
			<td>Band-aids</td>
			<td></td>
			<td></td>
			<td>Used for blood draw</td>
		</tr>
		<tr>
			<td>BD Bacto Tryptic Soy Broth</td>
			<td>BD</td>
			<td>DF0370-07-5</td>
			<td>Combine with 1.5% agar to make Tryptic Soy Agar</td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>Bal Saupply</td>
			<td>202C</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker blue CMCH</td>
			<td>Invitrogen</td>
			<td>C2111</td>
			<td>Blue CMAC Dye (BCD)</td>
		</tr>
		<tr>
			<td>Clear bottom 96-well flat bottom polystyrene plates</td>
			<td>Costar</td>
			<td>3370</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton gauze</td>
			<td>Fisherbrand</td>
			<td>13-761-52</td>
			<td>Used for blood draw</td>
		</tr>
		<tr>
			<td>Crystal violet</td>
			<td>Acros Organic</td>
			<td>40583-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture tubes</td>
			<td>Fisherbrand</td>
			<td>14-961-27</td>
			<td>Borosilicate Glass 13 x 100 mm</td>
		</tr>
		<tr>
			<td>D-(+)-glucose</td>
			<td>Sigma</td>
			<td>G-8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran from Leuconostoc spp.</td>
			<td>Sigma</td>
			<td>31392-250G</td>
			<td>Used for isolation of neutrophils</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS) 1x</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium homodimer-1</td>
			<td>Invitrogen</td>
			<td>L3224 B</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque plus</td>
			<td>Cytiva</td>
			<td>17144003</td>
			<td>Used for isolation of neutrophils (density gradient medium)</td>
		</tr>
		<tr>
			<td>Hanks&#39; balanced salt solution (HBSS) 1x</td>
			<td>Corning cellgro</td>
			<td>21-022-CV</td>
			<td>without calcium, magnesium, and phenol red</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Bright Line</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Novaplus</td>
			<td>NDC 63323-540-57</td>
			<td>1000 USP units/mL, Used for blood draw</td>
		</tr>
		<tr>
			<td>IMARIS 9.8</td>
			<td>Oxford Instruments</td>
			<td></td>
			<td>Microscopy image analysis software</td>
		</tr>
		<tr>
			<td>Luminol</td>
			<td>Sigma</td>
			<td>A8511-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimal essential media (MEM) Alpha 1x</td>
			<td>Gibco</td>
			<td>41061-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle (23 G1)</td>
			<td>BD</td>
			<td>305145</td>
			<td>Used for blood draw</td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti2</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Quantification of dead neutrophils</td>
		</tr>
		<tr>
			<td>Normal human serum</td>
			<td>Complement Technology</td>
			<td>NHS</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish (100 x 15 mm)</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine solution</td>
			<td>Sigma</td>
			<td>P4707-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Bioreagents</td>
			<td>BP358-10</td>
			<td>Used for neutrophil isolation</td>
		</tr>
		<tr>
			<td>SoftMax Pro Software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Microplate reader software used for data acquisition</td>
		</tr>
		<tr>
			<td>SpectraMax i3x</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Microplate reader</td>
		</tr>
		<tr>
			<td>Sterile water for irrigation, USP</td>
			<td>Baxter</td>
			<td>2F7114</td>
			<td>Endotoxin-free; Used for neutrophil isolation</td>
		</tr>
		<tr>
			<td>Surflo winged infusion set</td>
			<td>Terumo</td>
			<td>SC*19BLK</td>
			<td>19 G x 3/4&#34;, used for blood draw</td>
		</tr>
		<tr>
			<td>Trypan blue stain (0.4%)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Turnicate</td>
			<td></td>
			<td></td>
			<td>Used for blood draw</td>
		</tr>
		<tr>
			<td>UltraPure distilled water</td>
			<td>Invitrogen</td>
			<td>10977015</td>
			<td></td>
		</tr>
		<tr>
			<td>White opaque 96-well plates</td>
			<td>Falcon</td>
			<td>353296</td>
			<td>Tissue culture treated and flat bottom plate</td>
		</tr>
		<tr>
			<td>&#956;-Slide VI 0.4</td>
			<td>Ibidi</td>
			<td>80601</td>
			<td>&#956;-channel slide</td>
		</tr>
	</tbody>
</table>

standardized in vitro assays to visualize and quantify interactions between human neutrophils and staphylococcus aureus biofilms

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of infection where they use processes such as phagocytosis, production of reactive oxygen and nitrogen species (ROS, RNS, respectively), NETosis (neutrophil extracellular trap), and degranulation to kill microbes and resolve the infection. Interactions between neutrophils and planktonic microbes have been extensively studied. There have been emerging interests in studying infections caused by biofilms in recent years. Biofilms exhibit properties, including tolerance to killing by neutrophils, distinct from their planktonic-grown counterparts. With the successful establishment of both in vitro and in vivo biofilm models, interactions between these microbial communities with different immune cells can now be investigated. Here, techniques that use a combination of traditional biofilm models and well-established neutrophil activity assays are tailored specifically to study neutrophil and biofilm interactions. Wide-field fluorescence microscopy is used to monitor the localization of neutrophils in biofilms. These biofilms are grown in static conditions, followed by the addition of neutrophils derived from human peripheral blood. The samples are stained with appropriate dyes prior to visualization under the microscope. Additionally, the production of ROS, which is one of the many neutrophil responses against pathogens, is quantified in the presence of a biofilm. The addition of immune cells to this established system will expand the understanding of host-pathogen interactions while ensuring the use of standardized and optimized conditions to measure these processes accurately.

The media used to grow bacterial biofilms influence the survival of neutrophils. Different media were tested to reduce the effect of media alone on the viability of neutrophils for studying neutrophil-biofilm interactions (Figure 1). Bacterial growth media such as Tryptic Soy Broth minimizes the viability of neutrophils, such that ~60% of neutrophils are alive after a 30 min incubation period at 37 &#176;C with 5% CO2. Mammalian cell culture media, such as MEM&#945;, does not affect the viability of neutrophils and supports the growth of S. aureus biofilms. In fact, minimal media promotes robust growth of biofilms in other bacteria46,47.
To assess the effect of media on biofilm growth and variability in biofilm biomass quantification after washing the biomass to eliminate planktonic cells, an 18 h S. aureus biofilm was grown in a 96-well plate, with wells treated or untreated with poly-L-Lysine. A nutrient-rich (Tryptic Soy Broth (TSB)) and minimal (MEM&#945;) media were used as-is or supplemented with 2% glucose. The biofilm biomass stained with CV revealed that S. aureus biofilm grown in MEM&#945; supplemented with 2% glucose produced the most robust biofilm among all tested media (Figure 2A). Furthermore, biofilms grown in PLL pretreated wells containing MEM&#945; + 2% glucose showed less variability than biofilms in PLL-untreated wells containing MEM&#945; + 2% glucose. These biofilms showed less variability in quantification via CV assay35 and the CFU/mL when plated after precisely handling biofilms for biomass quantification. These biofilms contained, on average, 1 x 108 CFU/mL, as demonstrated by plating the biofilms in 3 separate days (Figure 2B). This number is useful in determining the number of neutrophils to add to the biofilms for neutrophil functionality assays.
To measure ROS production by neutrophils in response to biofilms, S. aureus biofilms were grown statically for 18-20 h in a 96-well plate. Biofilms were then opsonized, and neutrophils were added. ROS production was then measured for 60 min (Figure 3A). The area under the curve is calculated from the kinetic curve to quantify total ROS production by neutrophils. Neutrophils treated with an agonist, such as PMA, used as a control, show 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. In the absence of PMA, neutrophils solely rely on their interaction with the biofilm, which further reduces the amount of ROS produced (Figure 3B).
To visualize the neutrophil-biofilm interactions using fluorescence microscopy, a GFP-expressing strain of S. aureus, Blue CMAC dye, and ethidium homodimer-1, that stains the cytoplasm of live cells and DNA of dead cells, respectively, were used. S. aureus biofilm was grown for 18 h in a 6 &#181;-channel slide. Blue CMAC dye-labeled neutrophils were added along with ethidium homodimer-1 to the washed biofilms and incubated for 30 min at 37 &#176;C with 5% CO2 prior to imaging. Wide-field fluorescent microscopy revealed that many neutrophils were localized to the surface of S. aureus biofilms, while a few are within the biofilm (Figure 4A). The interaction between S. aureus cells within neutrophils was also apparent (Figure 4C). Most of the S. aureus cells interacting with neutrophils (cyan) were dead (magenta), while a few remained alive (yellow) as determined by live-dead staining (Figure 4C). For comparison, GFP-expressing S. aureus biofilms were stained with ethidium homodimer-1, which revealed a fraction of the dead S. aureus population within the biofilm (Figure 4B). Non-viable neutrophils that were positive for ethidium homodimer-1 were quantified using analysis software (see Table of Materials) after incubation with S. aureus biofilms. Approximately 48% of neutrophils were already dead within 30 min of incubation with S. aureus biofilm. During optimization of the microscopy protocol, the effect of washing the biofilm and neutrophils after 30 min of incubation to remove non-adhered neutrophils was also assessed, revealing around 33% of dead neutrophils still attached to the biofilm (Figure 4D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63773/63773fig1v2.jpg" /> 
Figure 1: LIVE-DEAD assay compares neutrophil survival between bacterial and mammalian growth media. Neutrophils were isolated and incubated in HBSS, MEM&#945;, TSB, or 0.1% SDS for 30 min. LIVE-DEAD staining was performed using Calcein AM (live) and ethidium homodimer-1 (dead). Percent of live neutrophils was determined, where HBSS-incubated neutrophils were treated as 100% live neutrophils. Results represent an average of two independent experiments performed in triplicate, with neutrophils obtained from two different donors. Data are presented as mean &#177; SD (*p &#60; 0.05, ****p &#60; 0.0001. One-way ANOVA). <a href="https://www.jove.com/files/ftp_upload/63773/63773fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63773/63773fig02.jpg" /> 
Figure 2: Quantification of biofilm biomass in different conditions and bacterial viability count of biofilms grown in the optimized conditions. (A) S. aureus was seeded in a 96-well plate either coated or uncoated with poly-L-Lysine (PLL). Biofilms were grown in TSB, MEM&#945;, or either of the media supplemented with 2% glucose under static conditions for 18 h. Crystal violet (CV) staining was performed to stain biofilm biomass. The eluted CV stain was diluted at 1:10 and read in a microplate reader. Results represent an average of three independent experiments performed in triplicate. Data are presented as mean &#177; SD. The SD for each group is shown at the bottom to demonstrate different biofilm growth conditions variability. (B) Bacterial CFU counts were obtained from biofilms grown in an optimized medium (MEM&#945; + 2% glucose). The 18 h static biofilms were subjected to the same number of washes followed by a 10 min sonication to loosen the biofilm biomass and passed through a 22G needle to disrupt the aggregates prior to plating. Results represent three replicates performed in triplicate. Data are presented as mean &#177; SD (ns = not significant. One-way ANOVA). <a href="https://www.jove.com/files/ftp_upload/63773/63773fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63773/63773fig03.jpg" /> 
Figure 3: Quantification of ROS production by neutrophils via chemiluminescence assay. (A) Neutrophils (PMN) were incubated with HBSS-washed S. aureus biofilms (BF) either in the presence (closed gray triangle) or absence (open gray inverted triangle) of PMA to measure ROS production by neutrophils. Luminol was used to detect ROS every 3 min for 60 min in a microplate reader. While neutrophils treated with PMA in the absence of a biofilm (closed black circle) served as a positive control, neutrophil only (open black circle) and biofilm only (open gray triangle) groups served as negative controls. Data represent an average of two independent experiments performed in triplicate with neutrophils obtained from two different donors. Data are presented as mean &#177; SD. (B) The area under the curve from (A) was calculated to quantify the total ROS generated by the neutrophils. The data are represented as mean &#177; SD. (***p &#60; 0.0001. One-way ANOVA). <a href="https://www.jove.com/files/ftp_upload/63773/63773fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63773/63773fig04.jpg" /> 
Figure 4: Visualization of the interaction between S. aureus biofilm and neutrophils using wide-field fluorescence microscopy. Blue CMAC dye-labeled neutrophils (cyan) were supplemented with ethidium homodimer-1 (magenta; dead) prior to incubating with an 18 h S. aureus biofilm (yellow). Biofilm-neutrophil interactions were imaged using wide-field fluorescent microscopy and images processed using an image analysis software. Experiments were performed with three different donors. Representative images are presented as (A) 3D view of S. aureus biofilm with live (cyan) and dead (magenta; a few indicated with white arrows) neutrophils, (B) 3D view of an S. aureus biofilm in the absence of neutrophils with either live S. aureus expressing GFP (yellow) or dead S. aureus stained with ethidium homodimer-1 (magenta), (C) an orthogonal view of S. aureus and neutrophil interaction as depicted by the xy, yz, and xz planes, and (D) quantification of neutrophil viability in the presence of S. aureus biofilm after 30 min either immediately (unwashed) or after three rounds of washes with HBSS to remove non-adhered neutrophils (washed). Neutrophil cell death is presented as mean &#177; SD (Student&#39;s t-test). Scale bar indicates 50 &#181;m in (A) and (B) and 10 &#181;m in (C). <a href="https://www.jove.com/files/ftp_upload/63773/63773fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms at JoVE.com

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

The present protocol describes the study of neutrophil-biofilm interactions. Staphylococcus aureus biofilms are established in vitro and incubated with peripheral blood-derived human neutrophils. The oxidative burst response from neutrophils is quantified, and the neutrophil localization within the biofilm is determined by microscopy.

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

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of ...

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JoVE Journal

Immunology and Infection

2.4K Views.  The Ohio State University. The present protocol describes the study of neutrophil-biofilm interactions. Staphylococcus aureus biofilms are established in vitro and incubated with peripheral blood-derived human neutrophils. The oxidative burst response from neutrophils is quantified, and the neutrophil localization within the biofilm is determined by microscopy. 

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

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...