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>

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	<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. 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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. 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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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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><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&#039;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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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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Nanyang Technological University

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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

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells&#39; mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes &#8211; Hoechst 33342 and propidium iodide.

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...

Cancer Research

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. ...

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

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.

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.

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

Optimized In Vivo Models for Intracellular S. aureus Infection Studies

Development and Assessment of Intracellular Infection Models for Staphylococcus aureus

S. aureus can invade and persist within host cells, including immune cells, which allows it to evade immune detection and clearance. This intracellular persistence contributes to chronic and recurrent infections, complicating treatment and prolonging the disease. Consequently, there is a critical need for an intracellular infection model to better understand, prevent, and treat infections caused by S. aureus. This study indicated that antibiotics effectively eliminated extracellular bacteria but could not eradicate those that had entered the cells. Thus, a stable intracellular infection in vitro was established by RAW264.7 infected with S. aureus and co-culturing them with antibiotics. Subsequently, an intracellular infection model in mice was established by injecting peritoneal macrophages containing the intracellular infection. Vancomycin effectively cleared bacterial loads in mice challenged with planktonic S. aureus; however, it was ineffective against mice infected with equal or lower levels of intracellular bacteria within the peritoneal macrophages. This indicates that the intracellular infection model of S. aureus was successfully established, offering potential insights for the prevention and treatment of intracellular infections.

Staphylococcus aureus (S. aureus) has the capability to disseminate throughout the body, causing persistent and recurrent infections. To better understand these processes, this study establishes an intracellular infection model for S. aureus. This model will provide a crucial foundation for investigating the mechanisms behind intracellular infections.

S. aureus can invade and persist within host cells, including immune cells, which allows it to evade immune detection and clearance. This intracellular ...

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. 
At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular ...

Biology

Calcein Acetoxymethyl Staining to Evaluate Natural Killer Cell-Mediated Cytotoxicity toward Cancer Cells

Source: Chava, S. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60714">Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells.</a> J. Vis. Exp. (2020)
This video demonstrates an assay to measure the cytotoxicity of natural killer cells against cancer cells using the Calcein AM stain. The reduced number of fluorescent cancer cells following co-culture with natural killer cells indicates natural killer cell-mediated cytotoxicity toward cancer cells.

Source: Chava, S. et al., Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

Lysostaphin-Based Enzyme Protection Assay: An In Vitro Method to Quantify Intracellular Staphylococcus aureus Load by Enzyme-Mediated Selective Killing of Extracellular Bacteria

Source: Rigaill, J. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62903">Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds.</a> J. Vis. Exp. (2021).
In this video, we demonstrate a lysostaphin-based in vitro enzyme protection assay to quantify the Staphylococcus aureus internalization in A549 cells. The assay helps in qualitative, quantitative, and characterization studies of the intracellular bacterial load in the host cells.

Source: Rigaill, J. et al. Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial ...

Biological Techniques

Assay Techniques

Cell-based assays

An Assay for Bacterial Extracellular Protein-Mediated Cytotoxicity on PMNs

Source: Dankoff, J. G., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60681">Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes.</a> J. Vis. Exp. (2020).
This video demonstrates an assay to investigate the cytotoxicity of proteins produced by Staphylococcus aureus, a pathogenic bacterium, on isolated human polymorphonuclear leukocytes (PMNs). The secreted cytotoxic proteins form pores on the PMN cell membrane, initiating cell death. Upon staining the dead cells using propidium iodide, flow cytometry is performed to quantify the dead cells and assess the cytotoxicity of the bacteria.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

Specialized Assays

Cytotoxicity Assay of Staphylococcus aureus Strains on Neutrophils following Phagocytosis

Source: Dankoff, J. G. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60681">Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes.</a> J. Vis. Exp. (2020)
In this video, we demonstrate an assay to detect the cytotoxicity of virulent and less virulent Staphylococcus aureus strains against human neutrophils following phagocytosis using flow cytometry.

1.&#160;S. aureus cytotoxicity assay against human polymorphonuclear leukocytes following phagocytosis
NOTE: Growth curves defined by the optical density ...

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

Summary

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Chapters in this video

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

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

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

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

Development and Assessment of Intracellular Infection Models for Staphylococcus aureus

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Calcein Acetoxymethyl Staining to Evaluate Natural Killer Cell-Mediated Cytotoxicity toward Cancer Cells

Lysostaphin-Based Enzyme Protection Assay: An In Vitro Method to Quantify Intracellular Staphylococcus aureus Load by Enzyme-Mediated Selective Killing of Extracellular Bacteria

An Assay for Bacterial Extracellular Protein-Mediated Cytotoxicity on PMNs

Cytotoxicity Assay of Staphylococcus aureus Strains on Neutrophils following Phagocytosis