<p class="jove_content"&gt; RN kısmen Avrupa Topluluğu Yedinci Çerçeve Programı, proje numarası 268324 Bir Marie Curie Avrupa Yeniden entegrasyon Hibeler (ERG) tarafından finanse edildi.</p&gt;

1. Yoğunluk Santrifüj tarafından İnsan Kanı itibaren PMNs&#39;nin izolasyonu <ol><li> Heparinize venöz kan 5 9 ml tüpler çizin. </li><li> Aşağıdaki gibi çift katmanlı Ficoll gradyanları (5 tüpler kan boyunca 4 gradyanları) hazırlanması: 50 ml&#39;lik bir tüp içinde bir yoğunluk 1.119 g / ml Ficoll çözeltisi, 12 ml ve üstüne bir yoğunluk 1.077 g / ml Ficoll çözeltisi dikkatli bir şekilde tabaka 10 ml dökün. </li><li> PBS eşit hacmi ile kan seyreltin. </li><li> Katman çift katmanlı Ficoll degrade dikkatle seyreltilmiş kan; degrade başına 20-25 ml. </li><li> Sallanan bir kova rotor, 22 ° fren olmadan C 396 x g&#39;de 20 dakika santrifüj. </li><li> Içeren soğuk RPMI hazırlanması% 0.05 insan serum albumini (HSA-içeren RPMI). Ayrıca 9 ml steril deiyonize H 2 O hazırlamak Ön serin buz üzerinde RPMI ve H 2 O hem. </li><li> (Steril bir pipet ucu koymak bir vakum pompasının kullanımı ile plazma (sarı renkli) ve PBMC, Ficoll içeren üst tabaka ve Ficoll ikinci tabaka (beyaz renkli) aspirePipet). </li><li> Küçük bir plastik pipet (her 2 degradelerin PMN kesirler için 1 tüp) kullanarak 50 ml tüpler PMN toplayın ve buz üzerine koyun. </li><li> 4 azından 249 x g&#39;de 10 dakika boyunca 50 ml santrifüj toplam hacim soğuk RPMI-HSA ekleme ° C </li><li> Bir vakum pompası ve vorteks yavaşça pelet (eritrositler ve PMN) kullanımı ile Süpernatantı al. </li><li> 9 ml steril deiyonize H 2 O ekleyin ve Sayacı başlatmak. 1 ml 10 kat konsantre PBS ekleyerek, tam 30 saniye sonra hiper ozmotik şok durdurun. Not: 30 saniye çok önemlidir. </li><li> 4 azından 249 x g&#39;de 10 dakika boyunca 50 ml santrifüj toplam hacim soğuk RPMI-HSA ekleme ° C </li><li> Bir yüksek vakum pompası kullanımı ile süpernatan çıkarmak ve tanımlanmış bir miktarda (1-2 mi) RPMI-HSA 1 tüp PMN pelet toplamak. </li><li> Hücrelerin miktarının belirlenmesi ve 1.10 7 hücre / ml konsantrasyonu ayarlanır. Donör bağlı olarak, izole PMNs verim Betwe olacakkan, her biri 9 ml tüp mesafede yer alan en 5 x 10 6, ve 3 x 10 7 PMN </li></ol> 2. İnsan PMN Kalsiyum Seferberlik Değerlendirme akışı Sitometrik Testi <ol><li> 2 uM Yük hücreleri (5 x 10 6 hücre / ml) Fluo-3-AM örneğin ışıktan korunan bir sallanan bir platform çalkalayıcı kullanılarak, oda sıcaklığında, 20 dakika sallama yavaş için RPMI-HSA ve inkübe. </li><li> Bu arada 10x son konsantrasyonda uyarıcı bir seri seyreltme (örneğin, 3-kat) hazırlanması. PSMα3 Bu protokolde kullanılan, ancak, nötrofiller üzerinde etki gösteren herhangi bir GPCR uyarıcı uygundur. </li><li> RPMI-HSA reseptörünün 10x konsantre edildi inhibitörü hazırlayın. Önleyicisi, oda sıcaklığında 10 dakika boyunca inhibitörü eşit hacmi ile uyarıcı (PSMα3 örn. HDL) önceden inkübe 25 ul uyarıcı görür. </li><li> Oda sıcaklığında, 249 x g&#39;de 10 RPMI-HSA ml santrifüj ekleyerek hücreleri yıkayın. 5 x tekrar süspansiyon hücreleri10 6 hücre / ml. </li><li> Sadece deneyden önce, 2x10 6 hücre / RPMI-HSA ml için hücreleri sulandırmak ve FACS tüp başına 200 ul hücreleri ekleyin. Seyreltilmiş PMN çok kırılgan. Bu konsantrasyonda çok uzun tutarak otomatik etkinleştirme yol açacaktır, aynı güçlü sallayarak veya pipetleme için de geçerlidir. </li><li> Akış sitometresinin için tüp takın, 3 saniye bekleyin ve satın alma başlar. Sabit bir süre (örneğin 8 sn) sonra, tüp çıkarmak ve hızlı bir şekilde örnek 50 ul uyarıcı ekleyin. Hemen numune tutucu üzerine tüp yeniden takın ve satın alma devam. </li><li> Uyaran en yüksek konsantrasyon ile en düşük ve bitiş ile başlayın. RPMI-HSA her çalıştırdıktan sonra düzenli akış sitometresi iğne yıkayın. </li><li> Akım sitometri analiz yazılımı ile verileri analiz. Uyaran eklenmesinden sonra bu için uyarıcı ilavesinden önce ortalama floresans sinyali karşılaştır ve AC hesaplamak için uygun bir pozitif ve negatif kontroller kullanmakölçülen her seyreltme için tivasyon gücü. </li></ol> Alternatif olarak, inhibitör reseptör inhibitör ile önceden inkübe hücrelerinde işlev görür. 3. PMN tarafından Fagositoz sonra Bakterilerin GFP İfade Floresan Mikroskopi Analizi <ol><li> S. büyütün suyu içinde gece boyunca ilgi muhabiri yapı (plazmid muhabiri korumak için gerekli antibiyotik) içeren aureus suşları. Bu durumda, yük MW2 PSMα bir GFP raportör yapı LB kültür ortamı, 5 ml, 50 ml&#39;lik plastik bir tüp içinde yetiştirilen, 10 kullanılır içeren içinde. </li><li> O / N ifadesi tüm GFP kaldırmak için, OD 660 0.01 kültür sulandırmak ve OD 660 0.1 büyüyecek. Bu kültürün 01:30 Redilute ve kültür 0.1 bir OD 660 ulaşana kadar büyüme izlemek. Santrifüj bakteri toplayın ve DPBS bir kez yıkayın. 1.0 OD 660 ya da R elde etmek için, orijinal hacminin 1:10 süspanseklişeler 5.10 8 CFU / ml. </li><li> 1.5 ml mikrotüp RPMI-HSA (oran 10:1) taze olarak izole edilen PMN (1.10 6 / ml) bakteriler (1.10 7 / ml) karıştırın ve% 10 arasında bir nihai konsantrasyona toplanmış insan serumu ekleyin. 37, 10 dakika boyunca bir çalkalama platformu üzerinde çalkalayın ° C fagositoz uyarmak için. 5.10 5 PMN / ml ve 8 de mermi kapak kayma bir kuyuya pipet 250 ul bakteri yüklü PMN sulandırmak. </li><li> Bir mikroskop bakteri ile görüntü PMN. Bir 40X/0.85 NA amacı ile donatılmıştır ters bir mikroskop iyi çalışıyor ve 37 stabil çevre tutmak için karanlık bir ortamda odasında kaplı olmalıdır ° C Kamera parlak bir alan ve zaman içinde GFP üretim takip GFP kanal hem de her 5-10 dk kullanarak önceden ayarlanmış konumları bir dizi için görüntüler elde. </li></ol>

<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. S. <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+Methicillin-Resistant+Staphylococcus+aureus:+Epidemiology+and+Clinical+Consequences+of+an+Emerging+Epidemic.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Formyl+Peptide+Receptor+2+Senses+Highly+Pathogenic+Staphylococcus+aureus.">Human Formyl Peptide Receptor 2 Senses Highly Pathogenic Staphylococcus aureus.</a> Cell Host &amp; Microbe. 7, 463 (2010).</li><li>Prat, 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=A+homolog+of+formyl+peptide+receptor-like+1+(FPRL1)+inhibitor+from+Staphylococcus+aureus+(FPRL1+inhibitory+protein)+that+inhibits+FPRL1+and+FPR.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Staphylococcal+Anti-Inflammatory+Protein+That+Antagonizes+the+Formyl+Peptide+Receptor-Like+1.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+inhibitory+protein+of+Staphylococcus+aureus,+a+bacterial+antiinflammatory+agent.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+Staphylococcal+Phenol+Soluble+Modulins+by+Serum+Lipoprotein+Particles.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Potential+New+Pathway+for+Staphylococcus+aureus+Dissemination:+The+Silent+Survival+of+S.+aureus+Phagocytosed+by+Human+Monocyte-Derived+Macrophages.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcal+alpha-phenol+soluble+modulins+contribute+to+neutrophil+lysis+after+phagocytosis.">Staphylococcal alpha-phenol soluble modulins contribute to neutrophil lysis after phagocytosis.</a> Cellular Microbiology. , (2013).</li><li>Queck, S. Y., 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=RNAIII-Independent+Target+Gene+Control+by+the+agr+Quorum-Sensing+System:+Insight+into+the+Evolution+of+Virulence+Regulation+in+Staphylococcus+aureus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+Bacterial+Imaging+In+Vivo.">Bioluminescent Bacterial Imaging In Vivo.</a> J. Vis. Exp. (69), e4318 (2012).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Burada açıklanan yöntemler bazı adımlar çok önemlidir. Biz burada bu vurgular. Yoğunluk dereceli santrifüj yoluyla nötrofil yalıtımı için santrifüj adımları sırasında ya da sonrasında katmanların rahatsız etmemek için önemlidir. Plastik bir pipet kullanarak nötrofil aspire zaman, hücre tabakasında ise, gibi çıkartma sıvı katmanları rahatsız edecek balon sıkmak için değil emin olun. Ayrıca görsel, ozmotik şok ve santrifüj aşamasından sonra topak, nöt...

Nötrofiller (PMN) Staphylococcus aureus 1 karşı doğuştan gelen bağışıklık yanıtı önemli bir rol oynamaktadır profesyonel fagositlerdir. Ev sahibi ve mikrop arasındaki sürekli bir savaş hem de bir silahlanma yarışı yol açmıştır. Metisilin dirençli S. son zamanlarda, toplum ile ilişkili (CA) suşları aureus (MRSA) nötrofil öldürme 2,3 tuzağa çok etkili gibi görünüyor ortaya çıkmıştır. CA-MRSA aşırı fenol çözünür modulin (PSM) üretimi 4,5 daha yüksek virulans ile ilişkilendirilmiştir. İnsan nötrofilleri reseptör 6 birleştiğinde Bu G-proteini ile aktive yol FPR2 ile bu PSM tanıyabilir. Ilk olaylardan biri kalsiyum hücre içi mağaza (Ca 2 +) harekete geçirilmesidir. Ca 2 + degranülasyonunun ve fagositoz 7 de dahil olmak üzere PMNs&#39;nin efektör çeşitli fonksiyonları için ikincil haberci olarak görev yapar. Bu nedenle Ca 2 + çok hassas bir göstergesidirPMN etkinleştirmek için PSM fonksiyonel kapasitesi. Nötrofiller üzerinde PSM etkilerini incelemek için, taze nötrofil kalsiyum mobilizasyonu üzerine fluoresces bir boya ile izole ve yüklenir. Bir akış sitometrisi deneyde bu hızlı harekete ölçülebilir. Bu yöntemi kullanarak, bu nötrofiller üzerindeki toksik ve diğer bileşenlerin doğrudan etkilerini incelemek ve bu aktif olan en düşük konsantrasyonunu belirlemek mümkündür. Bizim için, bu S. tarafından üretilen birçok proteinlerin etkisini araştırmak için çok yararlı bir araçtır aureus gibi FPR2 inhibitör protein (FLIPR) 8, 7 FLIPR benzeri, ve Staphylococcus aureus (CİPS) 9 Kemotaksis inhibitör protein gibi bağışıklık kaçırma, dahil. Tüm bu proteinler agonisti tanıma reseptöre bağlanarak nötrofillerde kalsiyum mobilizasyon inhibe ettiği gösterilmiştir. Son zamanlarda, bizim grup PSM işlevsel 10 serum lipoproteinlerin tarafından inhibe olduğunu nitelendirdi &lt;/&gt; Sup. Bu lipoproteinler PSM öncelikle hücre içi ortamda görevi yerine belirten, kan ve insan dokusu içinde bol miktarda mevcut bulunmaktadır. Kalsiyum mobilizasyon tahlilin durumu ki bu da serum çok düşük konsantrasyonları ile de inhibe ettiği gösterilen PSM tarafından nötrofil aktivasyonu, serum lipoproteinler etkisini ölçmek için izin verdi. PSM işlevsel serum tarafından inhibe bu yana, hücre içi toksinler gibi PSM için önemli bir işlevi olduğunu varsaydık. Bu nedenle fagositoz sonra PSM rolünü belirlemeyi amaçladık. Arası boşlukta PSM ifadesi araştırmak için, GFP psmα operon promotörünün raportör füzyonlarının inşa ettiler. Ne S. bu raportör suşları aureus nötrofiller tarafından fagosite edildi, ifade indüksiyon floresan mikroskobu 10 kullanılarak gözlemlenebilir oldu. Açıkçası, bu technique S. genin çok sayıda ifade çalışma sağlar fagositoz sonra aureus veya diğer patojenleri. S. beri arası niş hayatta aureus bu niş aktif genlerin rolü okuyan, doğuştan gelen bağışıklık sistemi 11 10 12 üstesinden gelmek için çok önemlidir onun virülans anlamak için büyük önem taşımaktadır.

<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

Biz nötrofiller üzerinde Staphylococcus aureus tarafından üretilen ve salgılanan fenol çözünür modulins (PSM) ve diğer toksinlerin etkisini araştırmak için yöntemler sunuyoruz. Biz yoğunluk gradiyenti santrifüj kullanılarak taze nötrofil izole nötrofiller üzerindeki PSM etkilerini incelemek. Bu nötrofil kalsiyum mobilizasyon üzerine fluoresces bir boya ile yüklenir. PSM tarafından nötrofil aktivasyonu serbest hücre içi kalsiyum konsantrasyonu hızlı ve geçici bir artış başlatır. Bir akış sitometrisi deneyde bu hızlı harekete serbest Ca2 + konsantrasyonunun artmasına tepki ön-yüklemeli bir boya floresan izlenmesi ile ölçülebilir. Bu yöntemi kullanarak, biz nötrofil aktive ve nötrofil aktivasyonu özel ve genel inhibitörlerinin etkilerini ölçmek için gerekli konsantrasyonu belirlemek PSM olabilir. Hücre içi alanı w PSM ifadesi araştırmake GFP için PSMα operonunun organizatörü muhabiri füzyon inşa ettik. Ne S. bu raportör suşları aureus nötrofiller tarafından fagosite edilir, ifade indüksiyon floresan mikroskobu kullanarak gözlemlenebilir.

Insan PMN kalsiyum seferberlik değerlendirilmesi için akış sitometrik analizi FL-1, sinyal bir artış ile gösterilir kalsiyum akısı ile ölçülen şekilde, hızlı aktivasyonu ile sonuçlanan sentetik PSMα3 bir konsantrasyon dizi nötrofil kuluçkalama ile hazırlanmıştır. % 0.01 sentetik PSMα3 ön inkübasyon,% 0.1 ya da% 1 insan serum önemli ölçüde kalsiyum akılarının (Şekil 1) ortaya çıkarma yeteneğini inhibe etmiştir. Flor...

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

Biz tarafından salgılanan PSM ve diğer toksinlerin etkisini araştırmak için yöntemler mevcut<em&gt; Staphylococcus aureus</em&gt; Akım sitometri ve floresan mikroskobu kullanarak nötrofiller üzerinde.

Etkileşimi incelenmesi<em&gt; Staphylococcus aureus</em&gt; Akım Sitometri ve Time Lapse Mikroskopi tarafından Nötrofiller ile

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

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

Research

JoVE Journal

Immunology and Infection

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

13.0K Görüntüleme.  University Medical Center Utrecht. Biz tarafından salgılanan PSM ve diğer toksinlerin etkisini araştırmak için yöntemler mevcut Staphylococcus aureus Akım sitometri ve floresan mikroskobu kullanarak nötrofiller üzerinde.

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

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

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

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

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

Damla Akış ve Döner Disk Reaktörler kullanın Staphylococcus aureus Biyofilm Analizi

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

İmmünoloji ve Enfeksiyon

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.

Subkutan Enfeksiyon Metisiline Dirençli Stafilokok 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 ...

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.

Demir Kaynağı Olarak İnsan Hemoglobin kullanarak Staphylococcus aureus Büyüme

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

Akım Sitometri tarafından İnsan Nötrofiller Nükleer Faktör Aktivasyon Algılama Basit ve Verimli Yöntemi

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Sitometrisi ile Primer İmmün hücre alt Floresan Nanoparçacık Etkileşimleri Algılama

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Real Time Anestetize Farelerde Intravital Mikroskopi Mezenterik Damarlardaki nötrofiller ve monositler Görüntüleme

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

bir Genotiplendirme<em&gt; Staphylococcus aureus</em&gt; Tarafından Ribozomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Yatay Gen Transferi Çalışmasında Metodolojisi<em&gt; Staphylococcus aureus</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Fagositoz fare makrofajlar tarafından hızlandırılmış 3D görüntüleme

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

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.

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.

Bir fare modeli için değerlendirmek doğuştan gelen bağışıklık yanıtı Staphylococcus aureus enfeksiyon

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