eoe sub articles

EoE Sub Articles

1. Preparation of in vitro biofilm
<ol>
	<li>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).</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 the 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. 
	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.</li>
</ol>
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.
<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, a 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 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 &#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 viability.</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>
3. 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), 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 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.</li>
</ol>

<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>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>Hemocytometer</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>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>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>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>

an in vitro assay to study the interaction between neutrophils and biofilm

Source: Rana, P. S. J. B., et al. <a href="https://app.jove.com/v/63773">Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms.</a> J. Vis. Exp. (2022)
This video demonstrates an in vitro assay to study neutrophil-biofilm interaction. The pathogenic bacteria embedded in the biofilm and isolated neutrophils were tagged using fluorescent labels, and the immune response by the neutrophils and the evasion of the immune response by the bacteria were assessed using a fluorescence microscope.

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

Source: Rana, P. S. J. B., et al. Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus ...

A biofilm is a community of surface-adhered bacteria embedded inside a matrix of extracellular polymeric substances, EPS, of microbial origin.
To study immune cell-biofilm interactions in vitro, take a slide with a channel containing a biofilm of Staphylococcus aureus &#8212; an opportunistic pathogen. The engineered bacteria express a fluorescent protein for microscopic detection. The channel connects to reservoirs filled with media to supply nutrients to the bacteria.
Add a suspension of neutrophils labeled with a fluorescent cell-tracking dye. The medium also contains an ethidium homodimer, which stains the dead cells&#39; DNA. Incubate under physiological conditions.
The pattern recognition receptors on neutrophils bind to the pathogen-associated molecular patterns on the bacteria. The binding induces neutrophils to phagocytose the bacteria and produce extracellular reactive oxygen species or ROS, killing the bacteria.
To evade the immune response, bacteria release detoxifying enzymes that diminish the ROS and leukocidin toxin that causes cell death. Monomeric leukocidin binds to specific receptors on the neutrophils and undergoes multimerization to form a pore, disrupting membrane integrity and killing the cells.
The ethidium homodimer enters through the damaged cell membrane, staining the dead cells.
Under a wide-field fluorescence microscope, a subset of bacterial cells and neutrophils appear dead, indicating neutrophil-biofilm interaction.

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Immunology

Immune Response

Host Defense Mechanism

81 Views. Source: Rana, P. S. J. B., et al. Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms. J. Vis. Exp. (2022) This video demonstrates an in vitro assay to study neutrophil-biofilm interaction. The pathogenic bacteria embedded in the biofilm and isolated neutrophils were tagged using fluorescent labels, and the immune response by the neutrophils and the evasion of the immune response by the bacteria were assessed using a f...

Video: An In Vitro Assay to Study the Interaction between Neutrophils and Biofilm - Concept

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

JoVE Journal

Immunology and Infection

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

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

Transcript

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

Clarifying and Imaging Candida albicans Biofilms

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms