Name: Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy (Video) | JoVE Journal
Uploaded: 2013-07-17T13:00:00.000+00:00
Duration: 10 min 40 s
Description: Learn to study Staphylococcus aureus interactions with human neutrophils using flow cytometry and time-lapse microscopy. This method enables analysis of bacterial toxin effects on immune cell activation in vitro.

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5 minutes

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>Centrifuge 20 min at 396 x g in a swinging bucket rotor, 22 &deg;C without braking.</li>
 <li>Prepare cold RPMI containing 0.05% Human Serum Albumin (RPMI-HSA). Also prepare 9 ml sterile deionized H2O. Pre-cool both the RPMI and H2O on ice.</li>
 <li>Aspirate the top Ficoll layer containing plasma (yellow colored) and PBMC, and the second layer of Ficoll (white colored) with the use of a vacuum pump (put a sterile pipette tip on the pipette).</li>
 <li>Collect the PMNs in 50 ml tubes using a small plastic pipette (1 tube for the PMN fractions of every 2 gradients), and place them on ice.</li>
 <li>Add cold RPMI-HSA to a total volume of 50 ml and centrifuge for 10 min at 249 x g at 4 &deg;C.</li>
 <li>Take off the supernatant with the use of a vacuum pump and vortex the pellet gently (erythrocytes and PMNs).</li>
 <li>Add 9 ml sterile deionized H2O and start the timer. Stop the hyper osmotic shock after 30 sec exactly, by adding 1 ml 10 times concentrated PBS. Note: the 30 sec are very critical.</li>
 <li>Add cold RPMI-HSA to a total volume of 50 ml and centrifuge for 10 min at 249 x g at 4 &deg;C.</li>
 <li>Take off the supernatant with the use of a vacuum pump and collect the PMN pellet in 1 tube with a defined volume (1-2 ml) RPMI-HSA.</li>
 <li>Determine the amount of cells and adjust the concentration to 1.107 cells/ml. Depending on the donor, the yield of isolated PMNs will be between 5 x 106 and 3 x 107 PMNs from each 9 ml tube of blood</li>
</ol>
2. Flow Cytometric Assay for Assessment of Calcium Mobilization in Human PMNs
<ol>
 <li>Load cells (5 x 106 cells/ml) with 2 &mu;M Fluo-3-AM in RPMI-HSA and incubate for 20 min at room temperature rocking slowly, using for instance a rocking platform shaker, protected from light.</li>
 <li>In the meantime prepare a serial dilution (e.g. 3-fold) of the stimulus at 10x the final concentration. PSM&alpha;3 is used in this protocol; however any GPCR stimulus which acts on neutrophils is suitable.</li>
 <li>Prepare 10x concentrated inhibitor of the receptor in RPMI-HSA. When the inhibitor acts on the stimulus (e.g. HDL on PSM&alpha;3) pre-incubate 25 &mu;l stimulus with an equal volume of inhibitor for 10 min at room temperature.</li>
 <li>Wash cells by adding 10 ml of RPMI-HSA and centrifuge for 249 x g at room temperature. Resuspend cells to 5 x 106 cells/ml.</li>
 <li>Just before the experiment, dilute the cells to 2x106 cells/ml in RPMI-HSA and add 200 &mu;l cells per FACS tube. Diluted PMNs are very fragile. Keeping them too long at this concentration will lead to auto-activation; the same holds for vigorous shaking or pipetting.</li>
 <li>Attach the tube to the flow cytometer, wait 3 sec and start acquisition. After a fixed time period (e.g. 8 sec), take off the tube and rapidly add 50 &mu;l stimulus to the sample. Immediately re-attach the tube onto the sample holder and continue the acquisition.</li>
 <li>Start with the lowest and end with the highest concentration of stimulus. Wash the flow cytometer needle regularly after each run with RPMI-HSA.</li>
 <li>Analyze data with flow cytometry analysis software. Compare the average fluorescence signal before addition of the stimulus to that after addition of the stimulus and use the appropriate positive and negative controls to calculate the activation strength for each dilution measured.</li>
</ol>
Alternatively when the inhibitor acts on the receptor pre-incubate the cells with the inhibitor.
3. Fluorescence Microscopy Analysis of Bacteria GFP Expression after Phagocytosis by PMNs
<ol>
 <li>Grow S. aureus strains containing a reporter construct of interest over night in broth (with antibiotics when required to maintain the reporter plasmid). In this case strain MW2 containing a PSM&alpha; GFP reporter construct is used 10, grown in a 50 ml plastic tube with 5 ml of LB culture medium.</li>
 <li>To remove all GFP from O/N expression, dilute the cultures to OD660 0.01 and grow to OD660 0.1. Redilute this culture 1:30, and monitor growth until the culture reaches an OD660 of 0.1. Collect the bacteria by centrifugation and wash once in DPBS. Resuspend in 1:10 of the original volume to obtain an OD660 of 1.0 or roughly 5.108 CFU/ml.</li>
 <li>Mix the bacteria (1.107/ml) with freshly isolated PMNs (1.106/ml) in RPMI-HSA (ratio 10:1) in a 1.5 ml microtube and add pooled human serum to a final concentration of 10%. Shake on a shaking platform for 10 min at 37 &deg;C to stimulate phagocytosis. Dilute the PMNs loaded with bacteria to 5.105 PMNs/ml and pipette 250 &mu;l into a well of an 8 well chambered cover slip.</li>
 <li>Image the PMNs with bacteria on a microscope. An inverted microscope equipped with a 40X/0.85 NA objective works well and should be encased in a dark environment chamber to keep the environment stably at 37 &deg;C. Acquire images for a number of preset positions using the camera every 5-10 min in both the bright field and the GFP channel to follow GFP production in time.</li>
</ol>

<ol><li>Rigby, K. M., DeLeo, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophils+in+innate+host+defense+against+Staphylococcus+aureus+infections%5BTitle%5D)+AND+%22Semin.+Immunopathol%22%5BJournal%5D)">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/pubmed?term=((Insights+into+Mechanisms+Used+by+Staphylococcus+aureus+to+Avoid+Destruction+by+Human+Neutrophils%5BTitle%5D)+AND+%22The+Journal+of+Immunology%22%5BJournal%5D)">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/pubmed?term=((Rapid+neutrophil+destruction+following+phagocytosis+of+Staphylococcus+aureus%5BTitle%5D)+AND+%22J.+Innate+Immun%22%5BJournal%5D)">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/pubmed?term=((Community-associated+meticillin-resistant+Staphylococcus+aureus%5BTitle%5D)+AND+%22The+Lancet%22%5BJournal%5D)">Community-associated meticillin-resistant Staphylococcus aureus.</a> The Lancet. 375, 1557(2010).</li>
<li>David, M. Z., Daum, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Community-Associated+Methicillin-Resistant+Staphylococcus+aureus%3A+Epidemiology+and+Clinical+Consequences+of+an+Emerging+Epidemic%5BTitle%5D)+AND+%22Clinical+Microbiology+Reviews%22%5BJournal%5D)">Community-Associated Methicillin-Resistant Staphylococcus aureus: Epidemiology and Clinical Consequences of an Emerging Epidemic.</a> Clinical Microbiology Reviews. 23, 616-687 (2010).</li>
<li>Kretschmer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+Formyl+Peptide+Receptor+2+Senses+Highly+Pathogenic+Staphylococcus+aureus%5BTitle%5D)+AND+%22Cell+Host+%26+Microbe%22%5BJournal%5D)">Human Formyl Peptide Receptor 2 Senses Highly Pathogenic Staphylococcus aureus.</a> Cell Host & Microbe. 7, 463(2010).</li>
<li>Prat, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+homolog+of+formyl+peptide+receptor-like+1+%28FPRL1%29+inhibitor+from+Staphylococcus+aureus+%28FPRL1+inhibitory+protein%29+that+inhibits+FPRL1+and+FPR%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">A homolog of formyl peptide receptor-like 1 (FPRL1) inhibitor from Staphylococcus aureus (FPRL1 inhibitory protein) that inhibits FPRL1 and FPR.</a> J. Immunol. 183, 6569-6578 (2009).</li>
<li>Prat, C., Bestebroer, J., de Haas, C. J. C., van Strijp, J. A. G., van Kessel, K. P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+New+Staphylococcal+Anti-Inflammatory+Protein+That+Antagonizes+the+Formyl+Peptide+Receptor-Like+1%5BTitle%5D)+AND+%22The+Journal+of+Immunology%22%5BJournal%5D)">A New Staphylococcal Anti-Inflammatory Protein That Antagonizes the Formyl Peptide Receptor-Like 1.</a> The Journal of Immunology. 177, 8017-8026 (2006).</li>
<li>de Haas, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemotaxis+inhibitory+protein+of+Staphylococcus+aureus%2C+a+bacterial+antiinflammatory+agent%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Medicine%22%5BJournal%5D)">Chemotaxis inhibitory protein of Staphylococcus aureus, a bacterial antiinflammatory agent.</a> The Journal of Experimental Medicine. 199, 687-695 (2004).</li>
<li>Surewaard, B. G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inactivation+of+Staphylococcal+Phenol+Soluble+Modulins+by+Serum+Lipoprotein+Particles%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">Inactivation of Staphylococcal Phenol Soluble Modulins by Serum Lipoprotein Particles.</a> PLoS Pathog. 8, e1002606(2012).</li>
<li>Kubica, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Potential+New+Pathway+for+Staphylococcus+aureus+Dissemination%3A+The+Silent+Survival+of+S.+aureus+Phagocytosed+by+Human+Monocyte-Derived+Macrophages%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages.</a> PLoS ONE. 3, e1409(2008).</li>
<li>Surewaard, B. G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Staphylococcal+alpha-phenol+soluble+modulins+contribute+to+neutrophil+lysis+after+phagocytosis%5BTitle%5D)+AND+%22Cellular+Microbiology%22%5BJournal%5D)">Staphylococcal alpha-phenol soluble modulins contribute to neutrophil lysis after phagocytosis.</a> Cellular Microbiology. , In Press (2013).</li>
<li>Queck, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNAIII-Independent+Target+Gene+Control+by+the+agr+Quorum-Sensing+System%3A+Insight+into+the+Evolution+of+Virulence+Regulation+in+Staphylococcus+aureus%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">RNAIII-Independent Target Gene Control by the agr Quorum-Sensing System: Insight into the Evolution of Virulence Regulation in Staphylococcus aureus.</a> Molecular Cell. 32, 150(2008).</li>
<li>Baban, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioluminescent+Bacterial+Imaging+In+Vivo%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Bioluminescent Bacterial Imaging In Vivo.</a> J. Vis. Exp. (69), e4318(2012).</li>
</ol>

The authors declare that they have no competing financial interests.

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><tbody><tr><td>Fluo-3, AM</td><td>Molecular Probes / Life Technologies</td><td>F-1241</td><td></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

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 PSMα3 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 PSMα3 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 phagocytosis by...

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. ...

The overall goal of the following experiment is to study the effects of staphylococcal phenol, soluble modlin or PSMs on human neutrophils. This is achieved by first isolating neutrophils from human blood for subsequent in vitro analysis. Next, the neutrophils are either loaded with fluorescent dye and mixed with a concentration series of PSMs with and without inhibitors, or they're allowed to phagocytose S reus bacteria containing A PSM alpha promoter GFP reporter construct.Ultimately activation of the neutrophils by PSM stimulation can be monitored by flow cytometry and expression of GFP by the phagocytosed. Bacteria can be analyzed by fluorescence microscopy By this method. You can study the effects of staphylococcal, phenyl, soy, molins, or neutrophils.Of course, this method can be applied to many other cell types, stimuli and antagonist. We first had the idea for the microscopy part of this method when discussing a way to observe the effects of PSMs inside neutrophils. Using a bacterial GFP expression construct and time-lapse microscopy allows us to do exactly that.After drawing five tubes of heparinized venous blood, pour 12 milliliters of a 1.119 grams per milliliter density fial solution into a 50 milliliter tube, and then carefully overlay 10 milliliters of a 1.077 grams per milliliter density FI call solution. Next, dilute the blood with an equal volume of PBS and layer 20 to 25 milliliters of blood carefully onto each dual layer fi call gradient. Centrifuge the gradients for 20 minutes at 396 times G and 22 degrees Celsius without breaking.And then use a vacuum pump fitted with a sterile pipette tip to aspirate the top yellow plasma and PBMC layers. And then the third white layer. Now use a small plastic pipette to transfer the polymorphonuclear neutrophil or PMN fraction from each tube into a new 50 milliliter tube.Add freshly prepared pre-cool our PMI supplemented with human serum albumin or R-P-M-I-H-S-A to each tube to bring the total volume to 50 milliliters. Then spin down the cells, aspirate the supernatant with a vacuum pump and gently vortex the red erythrocyte and PMN containing pellet. Next, add nine milliliters of sterile deionized water to the cells and start a timer.Add exactly 30 seconds. Add one milliliter of 10 times concentrated PBS to stop the hyperosmotic shock. Then wash the cells in a total volume of 50 milliliters of cold R-P-M-I-H-S-A.After removing the supernatant, add one milliliter of R-P-M-I-H-S-A to each palate, which is now white due to lysis of the erythrocytes. Then resus, suspend by pipetting and combine the pellets in one tube. Now count the cells and adjust the concentration to one times 10 to the seventh cells per milliliter.Now load five times 10 to the sixth cells per milliliter with two micromolar flu oh 3:00 AM in R-P-M-I-H-S-A. And cover the tube with aluminum foil to protect it from light. Next, incubate the cells for 20 minutes at temperature under agitation.In the meantime, prepare a threefold serial dilution of the stimulus at 10 times the final concentration. Then pre incubate 30 microliters of the stimulus with an equal volume of freshly prepared 10 times concentrated inhibitor or buffer for 10 minutes at room temperature. Next, wash the cells in 10 milliliters of R-P-M-I-H-S-A and resuspend the cells in fresh R-P-M-I-H-S-A at five times 10 to the sixth cells per milliliter.Right before flow analysis, dilute the cells to two times 10 to the sixth cells per milliliter in R-P-M-I-H-S-A and add 200 microliters of cells per fax tube. Being careful not to activate the cells by shaking or pipetting load the first tube on the flow cytometer. Wait three seconds and then start the acquisition after a fixed time period, for example, eight seconds, take off the tube and quickly add 50 microliters of the lowest dilution of the stimulus to the sample.Immediately reload the tube and continue the acquisition. When a dilution series is finished, wash the flow cytometer needle with fresh R-P-M-I-H-S-A before measuring a new series. After running the last sample, analyze the data with flow cytometry analysis software to analyze bacterial GFP expression.After PMN phagocytosis grow an S ous strain containing the reporter construct of interest overnight in broth, remove any GFP expressed during the overnight culture by diluting the cells to OD 6 60 0 0.01. Grow the culture to OD 6 60 0 0.1. Dilute the cells one to 30, and then monitor the growth until the culture once again reaches an OD six 60 of 0.1.Then collect the bacteria by centrifugation. Wash the cells once in DPBS and Resus. Suspend the culture.Add one to 10 of the original volume to obtain an OD six 60 of 1.0 or roughly five times 10 to the eighth CFU per milliliter. Now mix the bacteria with the freshly isolated PNS in R-P-M-I-H-S-A at a 10 to one ratio. Then add pooled human serum to a one 10th total end volume of pooled human serum within the cell solution.Stimulate phagocytosis by shaking the cell mixture for 10 minutes at 37 degrees Celsius. Then dilute the bacteria loaded PMNs to five times 10 to the fifth cells per milliliter, and transfer 250 microliters of the cells into each well of an eight well chambered cover slip. Finally, image the cells on an inverted microscope equipped with a 40 x objective and encased in a dark environment chamber and acquire images at multiple locations in the cover slip chambers every five to 10 minutes in both the bright field and the GFP channel to follow the bacterial GFP production in real time, incubating neutrophils with a concentration series of synthetic PSM alpha three results in a dose dependent rapid activation of the cells as measured by calcium flux.Pre incubation of the PSM alpha three peptide with 0.01%0.1%or 1%Human serum significantly inhibited the ability of the stimulus to elicit a calcium response. Again in a dose dependent fashion at 1%serum. Hardly any activation is visible at these PSM alpha three concentrations.In these images, two neutrophils containing phagocytose bacteria containing A PSM alpha GFP reporter construct are shown. The bacteria start to fluoresce about one hour after phagocytosis indicating expression of GFP from the PSM Alpha promoter. Bacteria outside the neutrophils do not fluoresce indicating that PSM alpha expression is rapidly switched on only when the bacteria are phagocytosed.When attempting this procedure is important to remember to always use fresh neutrophils for optimal results. After watching this video, I hope you have a good understanding of how to measure calcium flux by flow cytometry and how to measure bacterial gene expression inside eukaryotic cells using fluorescence microscopy.

studying-interactions-staphylococcus-aureus-with-neutrophils-flow

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Learn to study Staphylococcus aureus interactions with human neutrophils using flow cytometry and time-lapse microscopy. This method enables analysis of bacterial toxin effects on immune cell activation in vitro.

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

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