RN was partially funded by a Marie Curie European Re-integration Grants (ERG) from the European Community&#x2019;s Seventh Framework Programme, project number 268324.

1. Isolation of PMNs From Human Blood by Density Centrifugation
<ol>
 <li>Draw 5 9 ml tubes of heparinized venous blood.</li>
 <li>Prepare dual layer Ficoll gradients (4 gradients for 5 tubes blood) as follows: Pour 12 ml of a density 1.119 g/ml ficoll solution in a 50 ml tube and carefully layer 10 ml of a density 1.077 g/ml ficoll solution on top.</li>
 <li>Dilute the blood with an equal volume of PBS.</li>
 <li>Layer the diluted blood carefully on the dual layer Ficoll gradient; 20-25 ml per gradient.</li>
 <li>Cent...

<ol>
	<li>Rigby, K. M., 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=Neutrophils+in+innate+host+defense+against+Staphylococcus+aureus+infections.">Neutrophils in innate host defense against Staphylococcus aureus infections.</a> Semin. Immunopathol. 34, 237-259 (2012).</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> The Journal of Immunology. 175, 3907-3919 (2005).</li><li>Kobayashi, S. D., 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=Rapid+neutrophil+destruction+following+phagocytosis+of+Staphylococcus+aureus.">Rapid neutrophil destruction following phagocytosis of Staphylococcus aureus.</a> J. Innate Immun. 2, 560-575 (2010).</li><li>DeLeo, F. R., Otto, M., Kreiswirth, B. N., Chambers, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Community-associated+meticillin-resistant+Staphylococcus+aureus.">Community-associated meticillin-resistant Staphylococcus aureus.</a> The Lancet. 375, 1557 (2010).</li><li>David, M. Z., Daum, R. 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In the methods described here some steps are very critical. We will highlight these here.
For the isolation of neutrophils by density gradient centrifugation it is important not to disturb the layers during or after the centrifugation steps. When aspirating the neutrophils using a plastic pipette, make sure not to squeeze the balloon while in the layer of cells, as ejecting liquid will disturb the layers. Also, visually inspect the pellet of neutrophils after the osmotic shock and centrifugat...

Neutrophils (PMNs) are professional phagocytes that play a key role in the innate immune response against Staphylococcus aureus 1. The constant battle between host and microbe has led to an arms race of both. Recently, community-associated (CA) strains of meticillin resistant S. aureus (MRSA) have emerged that seem to be very efficient in circumvention of neutrophil killing 2,3. Excessive phenol soluble modulin (PSMs) production of CA-MRSA has been associated with higher virulence 4,5. Human neutrophils can recognize these PSMs via FPR2 which lead to activation of this G-protein coupled receptor 6. One of the earliest events is the mobilization of intracellular stores of calcium (Ca2+). Ca2+ acts as a secondary messenger for a variety of effector functions of PMNs including degranulation and phagocytosis 7. Therefore Ca2+ is a very sensitive indicator of the functional capacity of PSMs to activate PMNs. To study the effects of PSMs on neutrophils, fresh neutrophils are isolated and loaded with a dye that fluoresces upon calcium mobilization. In a flow cytometry experiment this rapid mobilization can be measured. Using this method, it is possible to study the direct effects of toxic and other components on neutrophils, and determine the lowest concentration at which these are active. For us, it is a very useful tool to study the effect of many proteins produced by S. aureus involved in immune evasion, such as FPR2 inhibitory protein (FLIPr) 8, FLIPr-like 7, and Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) 9. All these proteins have been shown to inhibit the calcium mobilization in neutrophils by binding to the receptor recognizing the agonist.
Recently, our group has described that PSMs are functionally inhibited by serum lipoproteins 10 . These lipoproteins are abundantly present within the blood and human tissue, indicating that PSMs exert their function primarily in the intracellular environment. The availability of the calcium mobilization assay allowed us to precisely measure the effect of serum lipoproteins on activation of neutrophils by the PSMs, indicated by the considerable inhibition by very low concentrations of serum.
Since the PSMs are functionally inhibited by serum, we hypothesized that there is an important function for PSMs as intracellular toxins. We therefore sought to determine the role of PSMs after phagocytosis. To investigate the expression of the PSMs in the intercellular space, we have constructed reporter fusions of the promoter of the psm&alpha; operon to GFP. When these reporter strains of S. aureus were phagocytosed by neutrophils, the induction of expression was observable using fluorescence microscopy 10. Obviously, this technique allows the study of the expression of a large number of genes in S. aureus or other pathogens after phagocytosis. Since for S. aureus surviving in the intercellular niche is very important to overcome the innate immune system11 10 12, studying the role of genes activated in this niche is highly relevant to understanding its virulence.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Fluo-3, AM</td>
			<td>Molecular Probes / Life Technologies</td>
			<td>F-1241</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ficoll-Paque</td>
			<td>GE Healthcare</td>
			<td>17-5442-03</td>
			<td>density 1.077 g/ml</td>
		</tr>
		<tr>
			<td>Histopaque</td>
			<td>Sigma</td>
			<td>11191</td>
			<td>density 1.119 g/ml</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco, Life Technologies</td>
			<td>52400-025</td>
			<td>contains 25 mM HEPES and L-glutamine</td>
		</tr>
		<tr>
			<td>Leica TCS SP5 microscope</td>
			<td>Leica Microsystems, The Netherlands</td>
			<td>TCS SP5</td>
			<td>objective: HCX PL APO 40X/0.85</td>
		</tr>
		<tr>
			<td>FACSCalibur</td>
			<td>BD Biosciences</td>
			<td>FACSCalibur</td>
			<td>Very important that the tube can be removed and replaced during the measurement process</td>
		</tr>
	</tbody>
</table>

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.

Flow cytometric assay for assessment of calcium mobilization in human PMNs
Incubating neutrophils with a concentration series of synthetic PSM&alpha;3 resulting in rapid activation as measured by calcium flux, which is shown by an increase in the signal in FL-1. Pre-incubation of synthetic PSM&alpha;3 with 0.01%, 0.1% or 1% human serum significantly inhibited the ability to elicit calcium fluxes (Figure 1).
Analysis of GFP expression in bacteria after pha...

Watch this Scientific Journal Video about Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy at JoVE.com

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.

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. ...

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Research

JoVE Journal

Immunology and Infection

13.0K visualizações.  University Medical Center Utrecht. Nós apresentamos métodos para estudar o efeito de PSMs e outras toxinas segregadas por Staphylococcus aureus Em neutrófilos utilizando citometria de fluxo e microscopia de fluorescência.