This work was supported by National Institutes of Health Grants R01 AI129302 (to S.I.S.) and the Training Program in Pharmacology: From Bench to Bedside at UC Davis (NIH T32 GM099608 to L.S.A). The Molecular and Genomic Imaging (CMGI) at the University of California Davis provided superb technological support.

All mouse studies were reviewed and approved by the Institutional Animal Care and Use Committee at UC Davis and were performed according to the guidelines of the Animal Welfare Act and the Health Research Extension Act. Be sure to use sterile gloves when working with animals.
1. Mouse Source and Housing
<ol>
	<li>Derive LysM-eGFP mice on a C57BL/6J genetic background as described previously12. Derive LysM-EGFP&#215;MyD88-/- mice by crossing LysM-EGFP mice with MyD88-/- mice on a C57BL/6J background.</li>
	<li>House mice in a vivarium. For these studies, animals were housed at the University of California, Davis in groups prior to surgery and single housed following surgery. Use mice between the ages of 10-16 weeks.</li>
</ol>
2. Bacterial Preparation and Quantification
<ol>
	<li>Remove the bioluminescent S. aureus strain of interest from -80 &#176;C storage to thaw on ice. Streak on a 5% bovine blood agar plate. Incubate the streaked plate in a humidified incubator at 37 &#176;C overnight (16 h). 
	NOTE: In this protocol, the ALC2906 SH1000 strain was used. This strain contains the shuttle plasmid pSK236 with the penicillin-binding protein 2 promoter fused to the luxABCDE reporter cassette from Photohabdus luminescens18.</li>
	<li>Prepare tryptic soy broth (TSB) by mixing 0.03 g of TSB powder per mL of pure water, and autoclave TSB to sterilize. When cooled, add any necessary antibiotics using sterile technique. In this protocol, add 10 &#181;g/mL of chloramphenicol18 to the TSB to select for expression of the pSK236 shuttle plasmid, which contains the bioluminescence luxABCDE cassette.</li>
	<li>Pick 3-4 separate colonies from the S. aureus plate into TSB with 10 &#181;g/mL chloramphenicol to start an overnight culture. Incubate bacteria on an incubating shaker at 37 &#176;C overnight (16 h).</li>
	<li>Start a new bacterial culture from the overnight culture by diluting a sample 1:50 into TSB with 10 &#181;g/mL chloramphenicol. Culture in an incubating shaker at 200 rpm and 37 &#176;C.</li>
	<li>Two hours after splitting the S. aureus, monitor the optical density at 600 nm (OD600) on a spectrophotometer. Observe the OD600 vs. time to find mid-logarithmic phase growth. For the ALC2906 SH1000 strain, an OD600 of 0.5 is mid-logarithmic and corresponds to a concentration of 1 x 108 CFU/mL (Figure 1).</li>
	<li>When OD600 is 0.5, wash bacteria 1:1 with ice-cold DPBS. Centrifuge the bacteria for 10 min at 3,000 x g and 4 &#176;C. Carefully decant the supernatant and add additional chilled DPBS and vortex thoroughly. Centrifuge once more for 10 min at 3,000 x g and 4 &#176;C.</li>
	<li>Carefully decant the supernatant. Resuspend the bacterial pellet at a desired concentration. For these studies, collect 3 mL of ALC2906 SH1000 and resuspend in 1.5 mL of PBS, correlating to a bacteria concentration of about 2 x&#160;108 CFU/mL. Keep bacteria on ice until use.</li>
	<li>To verify bacteria concentration, dilute 100 &#181;L of the bacterial sample 1:10,000 and 1:100,000 in PBS. Plate 20 &#181;L aliquots on an agar plate. Incubate at 37 &#176;C in a humidified incubator for 16 h. Count CFUs by gross examination and calculate a bacterial concentration the following day.</li>
</ol>
3. Excisional Skin Wounding and Inoculation with S. aureus
<ol>
	<li>Administer 100 &#181;L of 0.03 mg/mL buprenorphine hydrochloride (~0.2 mg/kg) to each mouse via intraperitoneal injection.&#160;</li>
	<li>Twenty minutes post-injection, place 2-4 mice in a chamber with 2-3 LPM oxygen with 2-4% isoflurane. Once mice are fully anesthetized, transfer the mice one at a time to a nose cone connected to 2-3 LPM oxygen with 2-4% isoflurane. Verify mice are fully anesthetized by firmly pinching each rear paw between a thumb and forefinger. Proceed to the next step if the animal does not respond to the pinch.</li>
	<li>Shave a 1 inch by 2 inch section on the back of the mouse with electric clippers and clear the area of fur clippings using a clean wipe or gauze. Avoid using depilatory lotion because it may cause excess inflammation.</li>
	<li>Clean the back of the mouse first with 10% povidone-iodine soaked gauze and then with a 70% ethanol soaked gauze. Clean the area in a spiral pattern, moving outward from the center of the surgical area. Wait approximately 1 min for the surgical area to dry prior to surgery.</li>
	<li>Hold the shaved back of the mouse loosely between two fingers and firmly press a sterile 6 mm punch biopsy at the center of the prepared surgical area. Do not pull the skin taut.
	<ol>
		<li>Twist the punch biopsy to create a circular outline on the skin that fully cuts through the skin in at least one section of the outline. Be careful not to cut into the underlying fascia or tissue.</li>
		<li>Use sterile scissors and forceps to cut through the epidermis and dermis following the circular pattern imprinted by the punch biopsy.</li>
	</ol>
	</li>
</ol>
4. S. aureus Inoculation
<ol>
	<li>Fill a 28 G insulin syringe with the desired bioluminescent bacterial inoculant. In this study, administer a concentration of 1 x&#160;108 CFU/mL (50 &#181;L). Do not administer more than 100 &#181;L of volume.</li>
	<li>Inject 50 &#181;L of inoculant between the fascia and tissue in the center of the wound on the back of the mouse. Ensure that the inoculant forms a bubble at the center of the wound with minimal leakage or dispersion.
	<ol>
		<li>Pull the dermis to the side, hold the syringe nearly parallel to the tissue, and slowly push the syringe into the tissue until a sudden decrease in resistance is felt, which indicates piercing of the fascia. Carefully lead the syringe into the center of the wound and dispense the inoculant slowly. Remove the syringe slowly from the animal.</li>
	</ol>
	</li>
	<li>Inject the same volume of sterile PBS into the wounds of uninfected animals as described above.</li>
	<li>Return the animal to its cage. Place the cage under a heat lamp or on top of a heating pad, and monitor the animal until recovery from anesthesia.</li>
</ol>
5. In Vivo BLI and FLI
<ol>
	<li>Initialize the whole animal imager through the instrument software. Anesthetize mice in a chamber receiving 2-3 LPM oxygen with 2-4% isoflurane. Deliver anesthesia to the nosecones inside the imager.</li>
	<li>Place the wounded and infected mouse into the imager. Position the mouse such that the wound is as flat as possible. Use the following sequence set-up to image the mice.
	<ol>
		<li>Select Luminescence and Photograph as the imaging mode. The exposure time is 1 min at small binning and F/stop 1 (luminescence) and F/stop 8 (photograph). The emission filter is Open. Click the Acquire button to record the image.</li>
		<li>Select Fluorescence and Photograph as the imaging mode. The exposure time is 1 s at small binning and F/stop 1 (Fluorescence) and F/stop 8 (photograph) with an excitation wavelength of 465/30 nm and an emission wavelength 520/20 nm with a high lamp intensity. Click the Acquire button to record the image.</li>
	</ol>
	</li>
	<li>Return the animal to its cage and monitor until recovery from anesthesia.</li>
	<li>Image mice daily as described above.</li>
</ol>
6. Image Analysis
<ol>
	<li>Open images to be quantified.</li>
	<li>Place a large circular region of interest (ROI) over the entire wound area including surrounding skin for each mouse in the image. The neutrophil in vivo FLI EGFP signals and in vivo BLI S. aureus signals extend beyond the wound edge after several days and were&#160;included in these studies (Figure 2A and 2B). Click Measure ROI and record values for mean flux for each mouse. Plot the mean flux of each signal (p/s) versus time.</li>
	<li>If absolute numbers of neutrophils or S. aureus in the wound are desired, perform the following experiments.
	<ol>
		<li>To correlate neutrophil numbers to the in vivo FLI EGFP signals, wound C57BL/6J mice as described above, and transfer a range of bone marrow-derived neutrophils (5 x 105 to 1 x 107) from LysM-EGFP or LysM-EGFPxMyD88-/- donors directly on top of different wounds. Image as described above and correlate the in vivo FLI EGFP signals to the known quantity of neutrophils.</li>
		<li>To correlate S. aureus CFU to the in vivo BLI signals, wound and infect mice as described above. On day 1 post-infection record in vivo BLI signals from the mice and immediately euthanize and chill carcasses. Excise the wound, homogenize the tissue, and plate bacterial dilutions on agar for overnight incubation. The next day, count colonies to determine CFU per wound.</li>
	</ol>
	</li>
	<li>To measure wound healing fit a circular ROI over the wound edge and measure the area of the wound (cm2) and plot the fractional change from baseline vs.&#160;time (Figure 2C).</li>
</ol>
7. Statistics
NOTE: All data are presented as mean &#177;SEM. p &#60; 0.05 were considered statistically significant
<ol>
	<li>Determine differences between two groups on a single day using the Holm-Sidak method, with alpha = 0.05, and analyze each time point individually, without assuming a consistent SD.</li>
	<li>Compare differences between multiple groups on the same day by one-way ANOVA with the Tukey multiple-comparisons posttest. Survival between experimental groups was analyzed by the Mantel-Cox method.</li>
</ol>

<ol>
	<li>Moran, G. J., 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=Methicillin-Resistant+S.+aureus+Infections+among+Patients+in+the+Emergency+Department.">Methicillin-Resistant S. aureus Infections among Patients in the Emergency Department.</a> New England Journal of Medicine. 355 (7), 666-674 (2009).</li><li>Suaya, J. A., 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=Incidence+and+cost+of+hospitalizations+associated+with+Staphylococcus+aureus+skin+and+soft+tissue+infections+in+the+United+States+from+2001+through+2009.">Incidence and cost of hospitalizations associated with Staphylococcus aureus skin and soft tissue infections in the United States from 2001 through 2009.</a> BMC Infectious Diseases. 14 (1), 296 (2014).</li><li>Ventola, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibiotic+resistance+crisis:+part+1:+causes+and+threats.">The antibiotic resistance crisis: part 1: causes and threats.</a> P &amp; T : a Peer-Reviewed Journal for Formulary Management. 40 (4), 277-283 (2015).</li><li>Blaser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+use+and+its+consequences+for+the+normal+microbiome.">Antibiotic use and its consequences for the normal microbiome.</a> Science. 352 (6285), 544-545 (2016).</li><li>Hilliard, J. J., 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=Anti-Alpha-Toxin+Monoclonal+Antibody+and+Antibiotic+Combination+Therapy+Improves+Disease+Outcome+and+Accelerates+Healing+in+a+Staphylococcus+aureus+Dermonecrosis+Model.">Anti-Alpha-Toxin Monoclonal Antibody and Antibiotic Combination Therapy Improves Disease Outcome and Accelerates Healing in a Staphylococcus aureus Dermonecrosis Model.</a> Antimicrobial Agents and Chemotherapy. 59 (1), 299-309 (2015).</li><li>Proctor, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+for+Staphylococcus+aureus+vaccines:+clinical+and+basic+science+challenges.">Recent developments for Staphylococcus aureus vaccines: clinical and basic science challenges.</a> European Cells &amp; Materials. 30, 315-326 (2015).</li><li>M&#246;lne, L., Verdrengh, M., Tarkowski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Neutrophil+Leukocytes+in+Cutaneous+Infection+Caused+by+Staphylococcus+aureus.">Role of Neutrophil Leukocytes in Cutaneous Infection Caused by Staphylococcus aureus.</a> Infection and Immunity. 68 (11), 6162-6167 (2000).</li><li>Kolaczkowska, E., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+recruitment+and+function+in+health+and+inflammation.">Neutrophil recruitment and function in health and inflammation.</a> Nature Reviews Immunology. 13 (3), 159-175 (2013).</li><li>Borregaard, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils,+from+Marrow+to+Microbes.">Neutrophils, from Marrow to Microbes.</a> Immunity. 33 (5), 657-670 (2010).</li><li>Miller, L. S., Cho, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+against+Staphylococcus+aureus+cutaneous+infections.">Immunity against Staphylococcus aureus cutaneous infections.</a> Nature Reviews Immunology. 11 (8), 505-518 (2011).</li><li>Hasenberg, A., 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=Catchup:+a+mouse+model+for+imaging-based+tracking+and+modulation+of+neutrophil+granulocytes.">Catchup: a mouse model for imaging-based tracking and modulation of neutrophil granulocytes.</a> Nature Methods. 12 (5), 445-452 (2015).</li><li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+green+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.</a> Blood. 96 (2), 719-726 (2000).</li><li>Falahee, P. C., 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=&#945;-Toxin+Regulates+Local+Granulocyte+Expansion+from+Hematopoietic+Stem+and+Progenitor+Cells+in+Staphylococcus+aureus-Infected+Wounds.">&#945;-Toxin Regulates Local Granulocyte Expansion from Hematopoietic Stem and Progenitor Cells in Staphylococcus aureus-Infected Wounds.</a> Journal of immunology. 199 (5), 1772-1782 (2017).</li><li>Kim, M. -. H., 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=Dynamics+of+Neutrophil+Infiltration+during+Cutaneous+Wound+Healing+and+Infection+Using+Fluorescence+Imaging.">Dynamics of Neutrophil Infiltration during Cutaneous Wound Healing and Infection Using Fluorescence Imaging.</a> Journal of Investigative Dermatology. 128 (7), 1812-1820 (2008).</li><li>Liese, J., Rooijakkers, S. H. M., Strijp, J. A. G., Novick, R. P., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+two-photon+microscopy+of+host-pathogen+interactions+in+a+mouse+model+of+Staphylococcus+aureus+skin+abscess+formation.">Intravital two-photon microscopy of host-pathogen interactions in a mouse model of Staphylococcus aureus skin abscess formation.</a> Cellular Microbiology. 15 (6), 891-909 (2013).</li><li>Bogoslowski, A., Butcher, E. C., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+recruited+through+high+endothelial+venules+of+the+lymph+nodes+via+PNAd+intercept+disseminating+Staphylococcus+aureus.">Neutrophils recruited through high endothelial venules of the lymph nodes via PNAd intercept disseminating Staphylococcus aureus.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (10), 2449-2454 (2018).</li><li>Takeuchi, O., Hoshino, K., Akira, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+Edge:+TLR2-Deficient+and+MyD88-Deficient+Mice+Are+Highly+Susceptible+to+Staphylococcus+aureus+Infection.">Cutting Edge: TLR2-Deficient and MyD88-Deficient Mice Are Highly Susceptible to Staphylococcus aureus Infection.</a> The Journal of Immunology. 165 (10), 5392-5396 (2000).</li><li>Miller, L. S., 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=MyD88+Mediates+Neutrophil+Recruitment+Initiated+by+IL-1R+but+Not+TLR2+Activation+in+Immunity+against+Staphylococcus+aureus.">MyD88 Mediates Neutrophil Recruitment Initiated by IL-1R but Not TLR2 Activation in Immunity against Staphylococcus aureus.</a> Immunity. 24 (1), 79-91 (2006).</li><li>Macedo, L., 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=Wound+healing+is+impaired+in+MyD88-deficient+mice:+a+role+for+MyD88+in+the+regulation+of+wound+healing+by+adenosine+A2A+receptors.">Wound healing is impaired in MyD88-deficient mice: a role for MyD88 in the regulation of wound healing by adenosine A2A receptors.</a> The American Journal of Pathology. 171 (6), 1774-1788 (2007).</li><li>Cho, J. S., 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=Neutrophil-derived+IL-1&#946;+Is+Sufficient+for+Abscess+Formation+in+Immunity+against+Staphylococcus+aureus+in+Mice.">Neutrophil-derived IL-1&#946; Is Sufficient for Abscess Formation in Immunity against Staphylococcus aureus in Mice.</a> PLoS Pathogens. 8 (11), e1003047 (2012).</li><li>Granick, J. L., 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+recognition+by+hematopoietic+stem+and+progenitor+cells+via+TLR2/MyD88/PGE2+stimulates+granulopoiesis+in+wounds.">Staphylococcus aureus recognition by hematopoietic stem and progenitor cells via TLR2/MyD88/PGE2 stimulates granulopoiesis in wounds.</a> Blood. 122 (10), 1770-1778 (2013).</li><li>Kim, M. H., 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=Neutrophil+survival+and+c-kit+-progenitor+proliferation+in+Staphylococcus+aureus-infected+skin+wounds+promote+resolution.">Neutrophil survival and c-kit+-progenitor proliferation in Staphylococcus aureus-infected skin wounds promote resolution.</a> Blood. 117 (12), 3343-3352 (2011).</li><li>Foster, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+evasion+by+staphylococci.">Immune evasion by staphylococci.</a> Nature Reviews Microbiology. 3 (12), 948-958 (2005).</li><li>Gordon, R. J., Lowy, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Methicillin-Resistant+Staphylococcus+aureus+Infection.">Pathogenesis of Methicillin-Resistant Staphylococcus aureus Infection.</a> Clinical Infectious Diseases. 46 (Supplement_5), S350-S359 (2008).</li><li>Cho, J. S., 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=Neutrophil-derived+IL-1&#946;+Is+Sufficient+for+Abscess+Formation+in+Immunity+against+Staphylococcus+aureus+in+Mice.">Neutrophil-derived IL-1&#946; Is Sufficient for Abscess Formation in Immunity against Staphylococcus aureus in Mice.</a> PLoS Pathogens. 8 (11), e1003047-e1003020 (2012).</li><li>Bernthal, N. 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=A+mouse+model+of+post-arthroplasty+Staphylococcus+aureus+joint+infection+to+evaluate+in+vivo+the+efficacy+of+antimicrobial+implant+coatings.">A mouse model of post-arthroplasty Staphylococcus aureus joint infection to evaluate in vivo the efficacy of antimicrobial implant coatings.</a> PLoS ONE. 5 (9), e12580 (2010).</li><li>Plaut, R. D., Mocca, C. P., Prabhakara, R., Merkel, T. J., Stibitz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stably+Luminescent+Staphylococcus+aureus+Clinical+Strains+for+Use+in+Bioluminescent+Imaging.">Stably Luminescent Staphylococcus aureus Clinical Strains for Use in Bioluminescent Imaging.</a> PLoS ONE. 8 (3), e59232 (2013).</li><li>Dillen, C. A., 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=Clonally+expanded+&#947;&#948;+T+cells+protect+against+Staphylococcus+aureus+skin+reinfection.">Clonally expanded &#947;&#948; T cells protect against Staphylococcus aureus skin reinfection.</a> The Journal of Clinical Investigation. 128 (3), 1026-1042 (2018).</li></ol>

Lloyd S. Miller has received grant support from MedImmune, Pfizer, Regeneron, and the Chan Soon-Shiong Nanthealth Foundation and consulting fees from Noveome Biotherapeutics and the Chan Soon-Shiong Nanthealth Foundation that are unrelated to the work reported in this paper. The other authors have nothing to disclose.

S. aureus infection models that utilize bioluminescent S. aureus infection in a fluorescent neutrophil reporter mouse in conjunction with advanced techniques of whole animal in vivo optical imaging have advanced our knowledge of the innate immune response to infection. Previous studies using the LysM-EGFP mouse have shown that up to 1 x 107 neutrophils recruit to an S. aureus infected wound over the first 24 h of infection14, and wound-recruited neutrophils ex...

Staphylococcus aureus (S. aureus) accounts for the majority of skin and soft tissue infections (SSTIs) in the United States1. The incidence of methicillin-resistant S. aureus (MRSA) infections has increased steadily over the past two decades2, motivating the study of the mechanisms of persistence and the discovery of new treatment strategies. The standard of care for MRSA infections is systemic antibiotic therapy, but MRSA has become increasingly resistant to antibiotics over time3 and these drugs can diminish the host&#39;s beneficial microbiome, causing negative health effects, especially in children4. Preclinical studies have examined alternative strategies to treat MRSA infections5, but translating these approaches to the clinic has proved challenging due to emergence of virulence factors that thwart host immune responses6. To dissect the host-pathogen dynamics that drive S. aureus SSTIs, we combine noninvasive and longitudinal readouts of the number of neutrophils recruited to the wound bed with kinetic measures of bacterial abundance and wound area.
Neutrophils are the most abundant circulating leukocyte in humans and the first responders to a bacterial infection7. Neutrophils are a necessary component for an effective host response against S. aureus infections due to their bactericidal mechanisms, including production of reactive oxygen species, proteases, antimicrobial peptides and functional responses including phagocytosis and neutrophil extracellular trap production8,9. Human patients with genetic defects in neutrophil function, such as chronic granulomatous disease and Chediak-Higashi syndrome, show an increased susceptibility to S. aureus infection. In addition, patients with genetic (such as congenital neutropenia) and acquired (such as neutropenia seen in chemotherapy patients) defects in neutrophil numbers are also highly susceptible to S. aureus infection10. Given the importance of neutrophils in clearing S. aureus infections, enhancing their immune capacity or tuning their numbers within a S. aureus lesion may prove an effective strategy in resolving infection.
Over the past decade, transgenic mice with fluorescence neutrophil reporters have been developed to study their trafficking11,12. Combining neutrophil reporter mice with whole animal imaging techniques permits spatiotemporal analysis of neutrophils in tissues and organs. When combined with bioluminescent strains of S. aureus, it is possible to track the accumulation of neutrophils in response to S. aureus abundance and persistence in the context of bacterial virulence factors that directly and indirectly perturb neutrophil numbers in affected tissue13,14,15,16.
Mice are less susceptible to S. aureus virulence and immune evasion mechanisms than humans. As such, wild-type mice may not be an ideal animal model to investigate the efficacy of a given therapeutic to treat chronic S. aureus infection. MyD88-deficient mice (i.e., MyD88-/- mice), an immunocompromised mouse strain that lacks functional interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR) signaling, show greater susceptibility to S. aureus infection compared to wild-type mice17 and an impairment in neutrophil trafficking to a site of S. aureus infection in the skin18. Development of a mouse strain that possesses a fluorescent neutrophil reporter in MyD88-/- mice has provided an alternative model for investigating the efficacy of therapies to treat S. aureus infection compared to current neutrophil reporter mice.
In this protocol, we characterize S. aureus infection in the immunocompromised LysM-EGFP&#215;MyD88-/- mice, and compare the time course and resolution of infection with the LysM-EGFP mice. LysM-EGFP&#215;MyD88-/- mice develop a chronic infection that does not resolve, and 75% succumb to infection after 8 days. A significant defect in initial neutrophil recruitment occurs over 72 h of the inflammatory phase of infection, and 50% fewer neutrophils recruit during the latter stage of infection. The increased susceptibility of the LysM-EGFP&#215;MyD88-/- mice makes this particular strain a rigorous preclinical model to evaluate the efficacy of new therapeutic techniques targeting S. aureus infection compared to current models that utilize wild-type mice, especially techniques aiming to boost the innate immune response against infection.

<table border="0" cellpadding="0" cellspacing="0" style="width:1101px;" width="1101">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">14 mL Polypropylene Round-Bottom Tube</td>
			<td style="width:301px;">Falcon</td>
			<td style="width:229px;">352059</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6mm Disposable Biopsy Punch</td>
			<td>Integra Miltex</td>
			<td>33-36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bioluminescent S. aureus</td>
			<td>Lloyd Miller, Johns Hopkins&#160;</td>
			<td>ALC 2906 SH1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Blood Agar, 5%, Hardy Diagnostics</td>
			<td>VWR</td>
			<td>10118-938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenoprhine hydrochloride injectable</td>
			<td>Western Medical Supply</td>
			<td>7292</td>
			<td>0.3 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6J Mice</td>
			<td>Jackson Labratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloramphenicol (crystalline powder)</td>
			<td>Fisher BioReagents</td>
			<td>BP904-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS (1X)</td>
			<td>Gibco&#160;</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin Syringes</td>
			<td>Becton, Dickson and Company</td>
			<td>329461</td>
			<td>.35 mm (28 G) x 12.7 mm (1/2&#39;&#39;)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IVIS Spectrum In Vivo Imaging System</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Living Image Software &#8211; IVIS Spectrum Series</td>
			<td>Perkin Elmer</td>
			<td>128113</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LysM-eGFP Mice</td>
			<td>Thomas Graff Albert Einstein College of New York&#160;</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microvolume Spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MyD88 KO Mice</td>
			<td>Jackson Labratory</td>
			<td>009088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-woven sponges</td>
			<td>AMD- Ritmed Inc</td>
			<td>A2101-CH</td>
			<td>5 cm x 5 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone Iodine 10% Solution</td>
			<td>Aplicare</td>
			<td>697731</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prism 7.0</td>
			<td>GraphPad Software</td>
			<td>License&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptic Soy Broth</td>
			<td>Becton, Dickson and Company</td>
			<td>211825</td>
			<td></td>
		</tr>
	</tbody>
</table>

a mouse model to assess innate immune response to staphylococcus aureus infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

LysM-EGFP&#215;MyD88-/- mice are more susceptible to S. aureus infection than LysM-EGFP mice
The strain of S. aureus used in this study, ALC290618, was constructed with a plasmid that contains the lux construct that produces bioluminescent signals from live and actively metabolizing bacteria. When inoculated into mice, in vivo bioluminescence imaging (BLI) techn...

Watch this Scientific Journal Video about A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection at JoVE.com

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

a-mouse-model-to-assess-innate-immune-response-to-staphylococcus

Research

JoVE Journal

Immunology and Infection

10.8K Views.  University of California Davis. An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Video: A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

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 ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

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.

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.

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

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...