This work was supported by the U.S. National Institutes of Health Grants NIH-1R56AI135039-01A1, 1R21A128295-01, U54GM115371 as well as funds from the Montana State University Agriculture Experiment Station, and an equipment grant from Murdock Charitable Trust.

Heparinized venous blood from healthy donors was collected in accordance with protocol approved by the Institutional Review Board for Human Subjects at Montana State University. All donors provided written consent to participate in this study.
1. Purification of human polymorphonuclear leukocytes and isolation of human serum
NOTE: All reagents should be routinely checked for the presence of endotoxin using a commercially available endotoxin detection kit and should contain &#60;25.0 pg/mL endotoxin to prevent unwanted priming of PMNs.
<ol>
	<li>Bring 50 mL of 3% dextran-0.9% NaCl (w/v), 35 mL of 0.9% NaCl (w/v), 20 mL of 1.8% NaCl (w/v), 12 mL of 1.077 g/mL density gradient solution, and 20 mL of injection- or irrigation-grade water to room temperature.</li>
	<li>To isolate human serum, incubate 4 mL of freshly drawn human blood without anti-coagulant at 37 &#176;C in a 15 mL glass tube for 30 min. After incubation, centrifuge sample at 2,000&#8211;3,000 &#215; g for 10 min at room temperature. Transfer the upper serum layer into a fresh 15 mL conical centrifuge tube and place on ice.</li>
	<li>Combine 25 mL of freshly drawn heparinized (1000 units/mL) whole human blood with 25 mL of room temperature 3% dextran-0.9% NaCl (1:1 ratio) in two replicate 50 mL conical centrifuge tubes (50 mL total volume per tube). Mix by gently rocking each 50 mL conical tube and then let stand at room temperature for 30 min.</li>
	<li>After incubation at room temperature, two separate layers will appear. Transfer the top layer of each dextran-blood mixture into new 50 mL conical tubes and centrifuge at 450 x g for 10 min at room temperature with low or no brakes.</li>
	<li>Carefully aspirate both supernatants and discard without disturbing the cell pellets. Gently resuspend each cell pellet in 2 mL of room temperature 0.9% NaCl, combine the resuspended pellets in a single 50 mL conical tube, then add the remaining 0.9% NaCl (final volume of 35 mL).</li>
	<li>Carefully underlay 10 mL of room temperature of 1.077 g/mL density gradient solution beneath the cell suspension using a hand pipette. Spin at 450 x g for 30 min at room temperature with low or no brakes. Gently aspirate the supernatant without disturbing the cell pellet. Supernatant will contain peripheral blood mononuclear cells that can be collected as previously described14.</li>
	<li>Lyse the red blood cells by resuspending the cell pellet in 20 mL of room temperature water. Mix gently by rocking the tube for 30 s. The lysis of red blood cells will be accompanied by a distinct decrease in turbidity.</li>
	<li>Immediately add 20 mL of 1.8% NaCl (at room temperature [RT]) and centrifuge sample at 450 &#215; g for 10 min at room temperature. 
	NOTE: It is important to minimize the time that PMNs are left in water alone following red blood cell lysis to maximize PMN yield and prevent PMN lysis and/or activation.</li>
	<li>Carefully aspirate the supernatant without disturbing the cell pellet. Gently resuspend the cell pellet in 2 mL of RT RPMI 1640 medium and place on ice.</li>
	<li>Count cells using a hemocytometer. Resuspend purified PMNs at a concentration of 1 x 107 cells/mL with ice-cold RPMI and keep on ice.</li>
	<li>Combine 100 &#181;L of purified PMNs (1 x 106 cells) with 300 &#181;L of ice-cold Dulbecco&#39;s phosphate-buffered saline (DPBS) containing 1 &#181;L of propidium iodide stain in two replicate flow cytometry tubes. For a positive control for plasma membrane damage, add 40 &#181;L of 0.5% Triton X-100 solution into one of the flow cytometry tubes and mix thoroughly.</li>
	<li>Use flow cytometry to measure the forward scatter, side scatter, and propidium iodide staining (excitation/emission maxima at 535/617 nm) of purified cells (Figure 1). 
	NOTE: Forward and side scatter analysis will identify unwanted populations of lymphocytes and monocytes. Propidium iodide will only stain cells with a compromised plasma membrane and purified PMNs that have pronounced populations of propidium iodide positive cells should not be used. For these studies, purified PMNs were only used if they comprised &#62;98% of purified cells and &#60;5% stained positive for propidium iodide.</li>
	<li>Prepare a 96-well plate for PMN cytotoxicity assays by coating individual wells that will be used in this assay with 100 &#181;L of 20% isolated human serum that has been diluted with DPBS. 
	NOTE: Plating PMNs directly on plastic or glass will cause activation of the cells. Be sure to include at least one negative control well that will only receive media and at least one positive control well that will receive 0.05% Triton X-100.</li>
	<li>Incubate the plate at 37 &#176;C for 30 min. Following incubation, wash the coated wells twice with ice-cold DPBS to remove any excess serum. Gently tap the plate upside down to remove any residual DPBS and place on ice.</li>
	<li>Gently add 100 &#181;L of purified human PMNs at 1 x 107 cells/mL to each coated well (1 x 106 PMNs/well). Allow PMNs to settle in wells by incubating the plate on ice for at least 5 min. Keep the plate level to allow even distribution of cells in each well and leave on ice to avoid unwanted activation of PMNs.</li>
</ol>
2. Cytotoxicity assay of S. aureus extracellular proteins against human polymorphonuclear leukocytes
<ol>
	<li>Culture S. aureus overnight in tryptic soy broth (TSB) using a shaking incubator set at 37 &#176;C. For these studies, 20 mL of TSB in separate 150 mL Erlenmeyer flasks were inoculated with frozen cultures of S. aureus strains USA300 or USA300&#916;saeR/S and grown for approximately 14 h with shaking at 250 rpm.</li>
	<li>Subculture S. aureus by performing a 1:100 dilution of overnight bacterial culture with fresh media. Incubate at 37 &#176;C with shaking until the bacteria reach early stationary growth phase. 
	NOTE: For these experiments, 20 mL of tryptic soy broth in 150 mL Erlenmeyer flasks were inoculated with 200 &#181;L of overnight cultured USA300 or USA300&#916;saeR/S and incubated at 37 &#176;C with shaking at 250 rpm for 5 h.</li>
	<li>When bacteria have reached early stationary growth phase, transfer 1 mL of subcultured S. aureus into a 1.5 mL microcentrifuge tube and centrifuge at 5,000 &#215; g for 5 min at room temperature.</li>
	<li>Following centrifugation, transfer supernatant into a 3 mL syringe. Pass supernatants through a 0.22 &#181;m filter and into a new 1.5 mL microcentrifuge tube on ice.</li>
	<li>Perform serial dilutions of supernatants with ice-cold media used to culture S. aureus. 
	NOTE: For the experiments shown, supernatants from USA300 and USA300&#916;saeR/S underwent four consecutive 1/2 log dilutions with ice-cold TSB.</li>
	<li>Gently add supernatant samples or media alone (for negative and positive controls) to individual wells of 96-well plate containing PMNs on ice from step 1.15. For these experiments, 10 &#181;L of USA300 or USA300&#916;saeR/S supernatant samples were added to each well. Gently rock plate to distribute supernatants in wells and incubate at 37 &#176;C.</li>
	<li>At desired times, remove the plate from incubator and place on ice. Add 40 &#181;L of 0.5% Triton X-100 to the positive control well.</li>
	<li>Gently pipette the samples up and down in each well to completely pull off all PMNs adhered to plate, then transfer the samples to flow cytometry tubes on ice that contain 300 &#181;L of ice-cold DPBS with 1 &#181;L of propidium iodide.</li>
	<li>Measure the proportion of propidium iodide-positive PMNs using flow cytometry (Figure 2A). When bound to DNA, propidium iodide has excitation/emission at 535/617 nm.</li>
</ol>
3. S. aureus cytotoxicity assay against human polymorphonuclear leukocytes following phagocytosis
NOTE: Growth curves defined by the optical density at 600 nm (OD600) and concentration of bacteria must be determined empirically for the S. aureus strains to be tested before beginning this assay. Success of these experiments requires the consistent harvest of equal concentrations of each S. aureus strain tested at mid-exponential growth phase using the OD600 of sub-cultured bacteria.
<ol>
	<li>Start overnight cultures of S. aureus strains and subculture bacteria as described in steps 2.1.1 and 2.1.2.</li>
	<li>Harvest subcultured S. aureus when it has reached mid-exponential growth by transferring 1 mL of cultured bacteria to a 1.5 mL microcentrifuge tube and centrifuging at 5,000 &#215; g for 5 min at room temperature. 
	NOTE: Under our growth conditions, USA300 and USA300&#916;saeR/S reached mid-exponential growth phase after approximately 135 min of incubation6.</li>
	<li>Wash S. aureus following centrifugation by aspirating the supernatant, resuspending the pelleted bacteria in 1 mL of DPBS, vortexing the sample for 30 s, and centrifuging at 5,000 &#215; g for 5 min at room temperature.</li>
	<li>Opsonize S. aureus by resuspending the bacterial pellet in 1 mL of 20% human serum diluted with DPBS and incubating at 37 &#176;C with agitation for 15 min.</li>
	<li>Centrifuge opsonized bacteria at 5,000 x g for 5 minutes at room temperature. Wash S. aureus following centrifugation by aspirating the supernatant, resuspending the pelleted bacteria in 1 mL DPBS, then vortex the sample until the bacterial pellet is completely broken apart plus an additional 30 seconds. Centrifuge bacteria at 5,000 &#215; g for 5 min at room temperature.</li>
	<li>Resuspend opsonized S. aureus strains in 1 mL RPMI, vortex the sample until bacterial pellet is completely broken apart, and then for an additional 30 s. Place bacteria on ice.</li>
	<li>Dilute opsonized S. aureus strains to the desired concentration with ice-cold RPMI. Vortex for 30 s and place on ice.</li>
	<li>Confirm the concentration of opsonized S. aureus by plating 1:10 serial dilutions of bacteria on tryptic soy agar. 
	NOTE: Because differences in the concentration of bacteria used in this assay can have a major impact on subsequent PMN plasma membrane permeability (Figure 3A), it is very important that the concentration of each strain tested is determined for every experiment and is equivalent between strains.</li>
	<li>Gently add 100 &#181;L/well of each S. aureus strain or RPMI (for positive and negative controls) to PMNs in the 96-well plate on ice from step 1.14. Gently rock plate to distribute S. aureus in wells.</li>
	<li>Synchronize phagocytosis by centrifuging the plate at 500 &#215; g for 8 min at 4 &#176;C15. Incubate plate at 37 &#176;C immediately following centrifugation (T = 0).</li>
	<li>At desired times, remove plate from incubator and place on ice. Add 40 &#181;L of 0.5% Triton X-100 to the positive control well.</li>
	<li>Gently pipette the samples up and down in each well to completely pull off all PMNs adhered to plate, then transfer the samples to flow cytometry tubes on ice containing 200 &#181;L of ice-cold DPBS with 1 &#181;L of propidium iodide.</li>
	<li>Analyze samples for propidium iodide staining using flow cytometry as described in step 2.9.</li>
</ol>

<ol>
	<li>Foster, T. J., Geoghegan, J. A., Ganesh, V. K., H&#246;&#246;k, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion,+invasion+and+evasion:+The+many+functions+of+the+surface+proteins+of+Staphylococcus+aureus.">Adhesion, invasion and evasion: The many functions of the surface proteins of Staphylococcus aureus.</a> Nature Reviews Microbiology. , (2014).</li><li>Thammavongsa, V., Kim, H. K., Missiakas, D., Schneewind, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcal+manipulation+of+host+immune+responses.">Staphylococcal manipulation of host immune responses.</a> Nature Reviews Microbiology. 13 (9), 529-543 (2015).</li><li>Otto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+toxins.">Staphylococcus aureus toxins.</a> Current Opinion in Microbiology. , (2014).</li><li>Guerra, F. E., Borgogna, T. R., Patel, D. M., Sward, E. W., Voyich, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epic+Immune+Battles+of+History:+Neutrophils+vs.+Staphylococcus+aureus.">Epic Immune Battles of History: Neutrophils vs. Staphylococcus aureus.</a> Frontiers in Cellular and Infection Microbiology. 7, (2017).</li><li>Nygaard, T., Malachowa, N., Kobayashi, S. D., DeLeo, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytes.">Phagocytes.</a> Management of Infections in the Immunocompromised Host. , 1-25 (2018).</li><li>Nygaard, T. K., Pallister, K. B., Ruzevich, P., Griffith, S., Vuong, C., Voyich, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SaeR+Binds+a+Consensus+Sequence+within+Virulence+Gene+Promoters+to+Advance+USA300+Pathogenesis.">SaeR Binds a Consensus Sequence within Virulence Gene Promoters to Advance USA300 Pathogenesis.</a> The Journal of Infectious Diseases. 201 (2), 241-254 (2010).</li><li>Voyich, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SaeR/S+gene+regulatory+system+is+essential+for+innate+immune+evasion+by+Staphylococcus+aureus.">The SaeR/S gene regulatory system is essential for innate immune evasion by Staphylococcus aureus.</a> J Infect Dis. 199 (11), 1698-1706 (2009).</li><li>Borgogna, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+Bacterial+Pneumonia+by+Staphylococcus+aureus+Following+Influenza+A+Infection+Is+SaeR/S+Dependent.">Secondary Bacterial Pneumonia by Staphylococcus aureus Following Influenza A Infection Is SaeR/S Dependent.</a> The Journal of Infectious Diseases. , (2018).</li><li>Guerra, F. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+SaeR/S-regulated+factors+reduce+human+neutrophil+reactive+oxygen+species+production.">Staphylococcus aureus SaeR/S-regulated factors reduce human neutrophil reactive oxygen species production.</a> Journal of Leukocyte Biology. 100, 1-6 (2016).</li><li>Zurek, O. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+innate+immunity+in+promoting+SaeR/S-mediated+virulence+in+Staphylococcus+aureus.">The role of innate immunity in promoting SaeR/S-mediated virulence in Staphylococcus aureus.</a> J Innate Immun. 6 (1), 21-30 (2014).</li><li>Nygaard, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspartic+Acid+Residue+51+of+SaeR+Is+Essential+for+Staphylococcus+aureus+Virulence.">Aspartic Acid Residue 51 of SaeR Is Essential for Staphylococcus aureus Virulence.</a> Frontiers in Microbiology. 9, 3085 (2018).</li><li>Spaan, A. N., Van Strijp, J. A. G., Torres, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocidins:+Staphylococcal+bi-component+pore-forming+toxins+find+their+receptors.">Leukocidins: Staphylococcal bi-component pore-forming toxins find their receptors.</a> Nature Reviews Microbiology. , (2017).</li><li>B&#248;yum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mononuclear+cells+and+granulocytes+from+human+blood.">Isolation of mononuclear cells and granulocytes from human blood.</a> Scandinavian Journal of Clinical and Laboratory. , (1968).</li><li>Nauseef, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+neutrophils+from+venous+blood.">Isolation of human neutrophils from venous blood.</a> Methods in Molecular Biology. , (2014).</li><li>Voyich, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+mechanisms+used+by+Staphylococcus+aureus+to+avoid+destruction+by+human+neutrophils.">Insights into mechanisms used by Staphylococcus aureus to avoid destruction by human neutrophils.</a> J Immunol. 175 (6), 3907-3919 (2005).</li><li>Voyich, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SaeR/S+gene+regulatory+system+is+essential+for+innate+immune+evasion+by+Staphylococcus+aureus.">The SaeR/S gene regulatory system is essential for innate immune evasion by Staphylococcus aureus.</a> The Journal of Infectious Diseases. 199 (11), 1698-1706 (2009).</li></ol>

The authors have nothing to disclose.

This protocol describes the purification of PMNs from human blood and two distinct assays that use propidium iodide for quantifying the cytotoxicity of S. aureus against these important innate immune cells. The success of these procedures will depend upon the quality of purified PMNs and the appropriate preparation of S. aureus and extracellular proteins produced by this pathogen. For the isolation of PMNs, it is important to minimize PMN activation during and after purification by using reagents free of endotoxin contamination, treating cell preparations gently, and keeping cells at the appropriate temperature. Signs that indicate activation of PMNs include clumping of cells during purification and when more than 5% of isolated cells stain positive for propidium iodide. Because of the relatively short life span of PMNs, these cells must be isolated from human blood and tested in the same day. PMNs will begin to exhibit signs of spontaneous apoptosis if left on ice for more than 3 h after purification. As mentioned earlier, it is very important that every PMN preparation is carefully evaluated using flow cytometry analysis of forward and side scatter as well as propidium iodide staining to ensure the purity and integrity of isolated cells.
The expression of bi-component leukocidins by S. aureus is responsible for the majority of compromised PMN plasma membrane integrity that is observed using the assays described in this protocol. Variation in the expression of these toxins and other pore-forming peptides, such as phenol-soluble modulins, between strains of S. aureus will produce differences in cytotoxicity against human PMNs. Significant deviations during in vitro growth between S. aureus strains will also influence expression of pore-forming toxins and subsequent cytotoxicity. In addition, the ratio of S. aureus to PMNs in phagocytosis assays has a major impact on subsequent PMN plasma membrane permeability (Figure 3A) and these experiments require the consistent harvest of equal concentrations of each S. aureus strain tested at mid-exponential growth phase using the OD600 of subcultured bacteria. Given these considerations, it is very important to define growth curves for all strains that will be examined before beginning cytotoxicity assays. We do not recommend these methods for analyzing S. aureus cytotoxicity with strains that exhibit significant growth differences in vitro.
USA300 is a virulent MRSA isolate that is known to be highly cytotoxic against human PMNs15 and the loss of SaeR/S in this strain dramatically reduces transcription of numerous bi-component leukocidins that target human PMNs6,12, making these strains ideal models for comparing cytotoxicity using the assays described. However, there is extensive genetic variation between different S. aureus isolates and the parameters detailed in these protocols may not result in substantial changes in cytotoxicity against human PMNs when testing other S. aureus strains. Tailoring the growth conditions, volumes of supernatants added, or ratio of bacteria to PMNs may be required for success with these methods using other strains of S. aureus.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/60681">Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes</a>. The title was&#160;updated.
The title was updated from:
Quantifying the Cytotoxicity of Staphyloccus&#160;aureus Against Human Polymorphonuclear Leukocytes
to:
Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

An erratum was issued for:&#160;<a href="https://www.jove.com/t/60681">Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes</a>. The title was&#160;updated.

Erratum: Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

Staphylococcus aureus is a Gram-positive bacterium that causes a wide spectrum of diseases in humans. This prominent pathogen produces numerous virulence factors that contribute to different aspects of infection. These include surface molecules that allow S. aureus to adhere to different types of host tissue1, extracellular proteins that interfere with the host immune response2, and an array of secreted toxins that target different types of host cells3. In this report, we describe a method that quantifies the cytotoxicity of extracellular proteins produced by S. aureus against human polymorphonuclear leukocytes (PMNs or neutrophils), primary effector cells of the host innate immune response.
PMNs are the most abundant leukocytes in mammals. These circulating immune cells are rapidly recruited to the site of host tissue insult in response to danger signals produced by resident cells or by compounds unique to invading microbes. The extracellular input from these molecules and from direct contacts with activated resident host cells during extravasation increase the activation state of PMNs in a process known as priming4,5. Primed PMNs that have reached distressed tissue then execute important innate immune responses designed to prevent the establishment of infection. These include the binding and internalization, or phagocytosis, of invading microorganisms that triggers a cascade of intracellular events cumulating in microbe destruction by a battery of potent antimicrobial compounds5.
PMNs play an essential role protecting humans from invading pathogens and are particularly important for preventing S. aureus infection4. However, this bacterium produces a wide range of virulence genes that impede different PMN functions. These include extracellular proteins that block recognition of signaling molecules, prevent adhesion to host tissue, inhibit production of antimicrobial compounds, and compromise plasma membrane integrity4. S. aureus orchestrates the temporal expression of these virulence genes through the collective input from multiple two-component sensory systems that recognize specific environmental cues. The SaeR/S two-component system is a major up-regulator of S. aureus virulence gene transcription during infection6,7,8,9,10,11. In particular, this two-component system has been shown to be critical for the production of bi-component leukocidins that specifically target human PMNs12.
This protocol is broken into three different sections. The first section describes the purification of PMNs from human blood using density gradient centrifugation using a protocol that has been adapted from methods established by B&#248;yum13 and Nauseef14. The second and third sections detail two different techniques to examine S. aureus cytotoxicity; one intoxicates PMNs with extracellular proteins produced by S. aureus while the other examines the ability of living bacteria to damage PMNs following phagocytosis. These procedures use propidium iodide to measure the loss of PMN plasma membrane integrity caused by S. aureus pore-forming toxins. Propidium iodide is a DNA-binding fluorophore that is normally cell membrane impermeable but can cross plasma membranes that have been disrupted by S. aureus toxins. Flow cytometry analysis allows the rapid quantification of propidium iodide-positive PMNs to measure the relative cytotoxicity of S. aureus strains. Methicillin-resistant S. aureus (MRSA) identified as pulsed-field gel electrophoresis type USA300 and an isogenic deletion mutant of saeR/S in this strain (USA300&#916;saeR/S) have been used as models to demonstrate how these procedures can quantify the cytotoxicity of S. aureus against human PMNs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% Sodium Chloride Injection, USP, 500 mL VIAFLEX Plastic Container</td>
			<td>Baxter</td>
			<td>2B1323Q</td>
			<td>PMN purification</td>
		</tr>
		<tr>
			<td>1.5 mL micro-centrifuge tubes with Snap Caps</td>
			<td>VWR</td>
			<td>89000-044</td>
			<td>Used in washing cells</td>
		</tr>
		<tr>
			<td>1.8% Sodium Chloride Solution</td>
			<td>Sigma-Aldrich</td>
			<td>S5150</td>
			<td>PMN purification</td>
		</tr>
		<tr>
			<td>12x75mm Culture tubes</td>
			<td>VWR</td>
			<td>60818-430</td>
			<td>Used as flow cytometry tubes</td>
		</tr>
		<tr>
			<td>20% (w/w) Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>D8802</td>
			<td>PMN purification</td>
		</tr>
		<tr>
			<td>3125 Hand Tally Counter</td>
			<td>Traceable Products</td>
			<td>3125CC</td>
			<td>For counting cells</td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tubes</td>
			<td>VWR</td>
			<td>89039-656</td>
			<td>For dispensing media</td>
		</tr>
		<tr>
			<td>Bacto Tryptic Soy Broth, Soybean-Casein Digest Medium</td>
			<td>FischerScientific</td>
			<td>211823</td>
			<td>For growing cell cultures</td>
		</tr>
		<tr>
			<td>BD Disposable Syringes with Luer-Lok Tips, 3 mL</td>
			<td>FischerScientific</td>
			<td>BD 309657</td>
			<td>For filtering supernatants</td>
		</tr>
		<tr>
			<td>Bright-Line Hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td>Cell counting apparatus</td>
		</tr>
		<tr>
			<td>DPBS, 1x (Dulbecco&#39;s Phosphate Buffered Saline) with calcium and magnesium</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td>Used in washing cells</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td>PMN purification</td>
		</tr>
		<tr>
			<td>Fisherbrand Sterile Polystyrene Disposable Serological Pipets</td>
			<td>FischerScientific</td>
			<td>13-678-11E</td>
			<td>For aspirating liquid</td>
		</tr>
		<tr>
			<td>Greiner CELLSTAR 96 well plates</td>
			<td>Millipore Sigma</td>
			<td>M0687</td>
			<td>Plate for holding experimental samples</td>
		</tr>
		<tr>
			<td>OMICRON Syringe Filters</td>
			<td>Omicron Scientific</td>
			<td>SFPV13R</td>
			<td>For filtering supernatants</td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>ThermoFisher Scientific</td>
			<td>P3566</td>
			<td>Membrane impermeable DNA stain</td>
		</tr>
		<tr>
			<td>PYREX Brand 4980 Erlenmeyer Flasks</td>
			<td>Cole-Parmer</td>
			<td>EW-34503-24</td>
			<td>For growing cell cultures</td>
		</tr>
		<tr>
			<td>RPMI 1640, 1X without L-glutamine, phenol red</td>
			<td>Corning</td>
			<td>17-105-CV</td>
			<td>Used in resuspending cells</td>
		</tr>
		<tr>
			<td>Sterile Water for Irrigation, USP</td>
			<td>Baxter</td>
			<td>2F7113</td>
			<td>PMN purification</td>
		</tr>
		<tr>
			<td>The Pipette Pump</td>
			<td>Bel-Art Products</td>
			<td>F37898</td>
			<td>For aspirating liquid</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td>Membrane integrity positive control</td>
		</tr>
	</tbody>
</table>

quantifying the cytotoxicity of staphylococcus aureus against human polymorphonuclear leukocytes

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes (PMNs or neutrophils). This protocol describes both the purification of human PMNs and the quantification of S. aureus cytotoxicity against PMNs in three different sections. Section 1 details the isolation of PMNs and serum from human blood using density centrifugation. Section 2 tests the cytotoxicity of extracellular proteins produced by S. aureus against these purified human PMNs. Section 3 measures the cytotoxicity against human PMNs following the phagocytosis of live S. aureus. These procedures measure disruption of PMN plasma membrane integrity by S. aureus leukocidins using flow cytometry analysis of PMNs treated with propidium iodide, a DNA binding fluorophore that is cell membrane impermeable. Collectively, these methods have the advantage of rapidly testing S. aureus cytotoxicity against primary human PMNs and can be easily adapted to study other aspects of host-pathogen interactions.

We have demonstrated how the procedures described above can be used to relatively quantify the cytotoxicity of S. aureus against human PMNs using MRSA PFGE-type USA300 and an isogenic deletion mutant of saeR/S in this strain (USA300&#916;saeR/S) generated in previous studies6. PMNs isolated using the procedures described in section 1 of this protocol were stained with propidium iodide and examined using flow cytometry. Forward and side scatter plots were used to illustrate contamination of purified PMNs by monocytes or lymphocytes (Figure 1A,B) and PMN integrity was determined using propidium iodide staining (Figure 1C). The described method of human PMN purification can consistently yield 0.5 x 107 to 1 x 108 PMNs that are &#62;98% pure and are &#62;95% propidium iodide negative.
The cytotoxicity of extracellular proteins produced by USA300 and USA300&#916;saeR/S were tested against purified PMNs (Figure 2) following the procedures described in section 2 of this protocol. These experiments demonstrate a concentration dependent increase in the propidium iodide staining of purified PMNs following 30 min of intoxication with extracellular proteins produced by USA300 (Figure 2B). Previous studies have demonstrated that the SaeR/S two-component system is important for expression of numerous bi-component leukocidins that target human PMNs6,10,11,16. Congruent with these previous findings, very few propidium iodide-positive PMNs were detected following exposure to extracellular proteins produced by USA300&#916;saeR/S (Figure 2B). Further experiments demonstrated a steady increase in the proportion of lysed PMNs following intoxication by USA300 extracellular proteins that plateaued after approximately 30 min (Figure 2C). Minimal lysis of human PMNs was noted at all timepoints following exposure to extracellular proteins produced by USA300&#916;saeR/S. These results illustrate the utility of this assay for the relative quantification of cytotoxicity by extracellular S. aureus proteins against human PMNs.
We tested USA300 and USA300&#916;saeR/S using the S. aureus cytotoxicity assay against human PMNs following phagocytosis that is described in section 3 of this protocol (Figure 3). A concentration dependent increase in the proportion of propidium iodide positive PMNs was observed 90 min after the phagocytosis of USA300 (Figure 3A). A significant decrease was observed in the proportion of PMNs that were propidium iodide positive following the phagocytosis of USA300&#916;saeR/S (Figure 3A), supporting other results that indicate the SaeR/S two-component system is important for the cytotoxicity of S. aureus against human PMNs (Figure 2)7,11. As previously mentioned and demonstrated in Figure 3A, differences in S. aureus concentration have a pronounced impact on PMN lysis following phagocytosis. Enumeration of the USA300 and USA300&#916;saeR/S inoculum used in each of these experiments demonstrated that the contrast in cytotoxicity between these strains was not due to differences in the concentration of bacteria used (Figure 3B). These findings show how the S. aureus cytotoxicity assay against human PMNs following phagocytosis can be used to assess the ability of different S. aureus strains to compromise human PMN plasma membrane integrity.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60681/60681fig1.jpg" /> 
Figure 1: Flow cytometry analysis of purified PMNs. Representative flow cytometry dot plots of (A) purified human PMNs and (B) PMNs that have been purposely contaminated with peripheral blood mononuclear cells. (C) Representative flow cytometry histogram demonstrating minimal propidium iodide staining (&#60;1%) of purified PMNs (shaded grey) as compared to PMNs treated with 0.05% Triton X-100 (shaded red). <a href="https://www.jove.com/files/ftp_upload/60681/60681fig1alarge.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/60681/60681fig2a.jpg" /> 
Figure 2: Flow cytometry analysis of PMNs intoxicated with extracellular proteins produced by S. aureus. (A) Representative flow cytometry histogram of PMNs stained with propidium iodide after 30 min of incubation with media control (shaded blue), filtered USA300 supernatant at a final concentration of 1:110 (shaded grey), or 0.05% Triton X-100 (shaded red). (B) The proportion of propidium iodide positive PMNs after 30 min of incubation with different concentrations of USA300 or USA300&#8710;saeR/S supernatants. (C) The proportion of propidium iodide positive PMNs over time following incubation with USA300 or USA300&#8710;saeR/S supernatant at a final concentration of 1:110. Data are presented as mean &#177; SEM of at least 3 separate experiments with * p &#8804; 0.05 and ** p &#8804; 0.005 as determined by two-tailed t-test. <a href="https://www.jove.com/files/ftp_upload/60681/60681fig2alarge.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/60681/60681fig3.jpg" /> 
Figure 3: Flow cytometry analysis of PMNs following phagocytosis of S. aureus. (A) The proportion of propidium iodide positive PMNs 90 min after the phagocytosis of different concentrations of USA300 or USA300&#8710;saeR/S. (B) Concentration of opsonized S. aureus strains used for the experiments shown in panel A. Data are presented as mean &#177; SEM of 4 separate experiments with * p &#8804; 0.01 as determined by two-tailed t-test. <a href="https://www.jove.com/files/ftp_upload/60681/60681fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes at JoVE.com

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

This protocol describes a method for the purification of polymorphonuclear leukocytes from whole human blood and two distinct assays that quantify the cytotoxicity of Staphylococcus aureus against these important innate immune cells.

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes ...

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Immunology and Infection

6.1K Views.  Montana State University. This protocol describes a method for the purification of polymorphonuclear leukocytes from whole human blood and two distinct assays that quantify the cytotoxicity of Staphylococcus aureus against these important innate immune cells.

Video: Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

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 ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

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. ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

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, ...