<p class="jove_content"&gt; RN 부분적으로 유럽 연합의 일곱 번째 프레임 워크 프로그램, 프로젝트 번호 268324에서 마리 퀴리 유럽의 재 통합 보조금 (ERG)에 의해 재정 지원되었다.</p&gt;

1. 밀도 원심 분리하여 인간의 혈액에서 PMNs의 분리 <ol><li> 헤파린 정맥 혈액의 3.5 9 ML 튜브를 그립니다. </li><li> 다음과 같이 듀얼 레이어 Ficoll 그라디언트 (5 관 혈액 4 그라디언트) 준비 : 50 ML 튜브의 밀도 1.119 g / ㎖ ficoll 용액 12 mL를 상단에 밀도 1.077 g / ㎖ ficoll 솔루션 조심스럽게 층을 10 ㎖를 붓고. </li><li> PBS의 동일한 볼륨 혈액을 희석. </li><li> 레이어 듀얼 레이어 Ficoll 기울기에주의 희석 된 혈액, 그라데이션 당 20-25 ML. </li><li> 스윙 버킷 회, 22 ° 제동없이 C에서 396 XG에서 20 분 원심 분리기. </li><li> 포함하는 차가운 RPMI를 준비 0.05 % 사람 혈청 알부민 (RPMI-HSA). 또한 9 ML 살균 이온 H 2 O를 준비 전 차가운 얼음에 RPMI와 H 2 O 양. </li><li> (멸균 피펫 팁을에 넣어 진공 펌프의 사용과 혈장 (노란 색)과 PBMC를 포함하는 상위 Ficoll 계층 및 Ficoll의 두 번째 계층 (화이트 컬러)를 대기음피펫). </li><li> 작은 플라스틱 피펫 (매 2 그라디언트 PMN 분수 1 관)을 사용하여 50 ML 튜브에 PMNs를 수집하고 얼음에 배치합니다. </li><li> 4에서 249 XG에서 10 분 동안 50 ML과 원심 분리기의 총 볼륨 차가운 RPMI-HSA를 추가 ° C. </li><li> 진공 펌프와 소용돌이 부드럽게 펠렛 (적혈구 및 PMNs)의 사용으로 상층 액을 제거한다. </li><li> 9 ML 살균 이온 H 2 O를 추가하고 타이머를 시작합니다. 1 ML 10 배 농축 PBS를 추가하여, 정확히 30 초 후 하이퍼 삼투 충격을 중지합니다. 참고 : 30 초는 매우 중요하다. </li><li> 4에서 249 XG에서 10 분 동안 50 ML과 원심 분리기의 총 볼륨 차가운 RPMI-HSA를 추가 ° C. </li><li> 진공 펌프의 사용과 상층 액을 가지고 정의 된 볼륨 (1-2 ML) RPMI-HSA와 1 관에서 PMN 펠렛을 수집합니다. </li><li> 세포의 양을 결정하고 1.10 7 세포 / ㎖로 농도를 조정합니다. 기증자에 따라 격리 PMNs의 수율은 betwe을 것입니다혈액의 각 10 ML 튜브에서 EN 5 × 10 6, 3 × 10 7 PMNs </li></ol> 2. 인간 PMNs에서 칼슘 동원의 평가를위한 유세포 분석 <ol><li> 2 μM과 짐 세포 (5 × 10 6 세포 / ML) 플루오-3-AM 예를 들어 빛으로부터 보호 락을 플랫폼 쉐이커를 사용하여, 실내 온도 요동에서 20 분 천천히 대한 RPMI-HSA와 부화한다. </li><li> 반면에 배 최종 농도 자극의 연속 희석 (예를 들어 3 배)를 준비. PSMα3이 프로토콜에서 사용되지만 백혈구에 작용하는 GPCR 자극이 적당하다. </li><li> RPMI-HSA에있는 수용체의 10 배 집중 억제를 준비합니다. 억제제는 실온에서 10 분 동안 억제제의 동일한 볼륨 자극 (PSMα3에 예 HDL) 사전 부화 25 μL 자극에 작용합니다. </li><li> 상온에서 249 XG에 대해 10 RPMI-HSA ml의 원심 분리기를 추가하여 세포를 씻으십시오. 5 X에 세포를 resuspend10 6 세포 / ML. </li><li> 그냥 실험을하기 전에, 2 × 10 6 세포 / RPMI-HSA에 ML로 세포를 희석 FACS 튜브 당 200 μl의 세포를 추가합니다. 희석 PMNs은 매우 허약하다. 이 농도에서 너무 오래 그들을 유지하는 것은 자동 활성화로 이어질 것입니다, 같은 격렬한 흔들림이나 피펫 팅을 위해 보유하고 있습니다. </li><li> 흐름 cytometer에에 튜브를 연결, 3 초를 기다렸다가 수집을 시작합니다. 고정 기간 (예 : 13 초) 한 후, 튜브를 타고 빠른 속도로 샘플 50 μL 자극을 추가합니다. 즉시 샘플 홀더에 튜브를 다시 연결하고 인수를 계속합니다. </li><li> 자극의 높은 농도와 가장 낮은과 끝 시작합니다. RPMI-HSA 각 실행 후 정기적으로 흐름 cytometer에 바늘을 씻으십시오. </li><li> 유동 세포 계측법 분석 소프트웨어를 사용하여 데이터를 분석 할 수 있습니다. 자극의 추가 후에 그에게 자극뿐만 아니라 이전의 평균 형광 신호를 비교하고 교류를 계산하기위한 적절한 긍정적이고 부정적인 컨트롤을 사용하여측정 된 각 희석 tivati​​on 강도. </li></ol> 또는 억제제 억제제 수용체 사전 부화 세포에 작용합니다. 3. PMNs에 의해 탐식 후 박테리아 GFP 발현의 형광 현미경 분석 <ol><li> S. 성장 국물에 밤에 그 기자 구문을 (플라스미드 기자를 유지하는 데 필요한 항생제)를 포함 구균의 변종. 이 경우 변형 MW2 PSMα GFP 기자 구조는 LB 배양액 5 ml의 50 ML 플라스틱 관에서 자란 10 사용된다 포함한다. </li><li> O / N 표현식에서 모든 GFP를 제거하려면, OD 660 0.01 문화를 희석 OD 660 0.1 성장합니다. 이 문화 1:30 Redilute, 그리고 문화 0.1의 OD 660에 도달 할 때까지 성장을 모니터링 할 수 있습니다. 원심 분리하여 박테리아를 수집하고 DPBS에 한 번 씻는다. OD 1.0 660 또는 R를 얻기 위해 원본 볼륨의 1:10 resuspend을하여 깨끗이 5.10 8 CFU / ㎖. </li><li> 1.5 ML의 모세관에있는 RPMI-HSA (비 10시 1분)에서 갓 고립 PMNs (1.10 6 / ML)와 박테리아 (1.10 7 / ㎖) 혼합 및 10 %의 최종 농도에 풀링 된 혈청을 추가합니다. 37 10 분 동안 흔들어 플랫폼에 흔들 ° C 식균 작용을 촉진합니다. 5.10 5 PMNs / ㎖와 8 잘 챔버 커버 슬립의 잘 피펫 250 μL에 세균로드 PMNs를 희석. </li><li> 현미경 세균 이미지 PMNs합니다. 40X/0.85 NA의 목적으로되어 거꾸로 현미경은 잘 작동하고 37 안정적 환경을 유지하기 위해 어두운 환경 챔버에 넣어해야 ° C. 카메라에게 밝은 필드와 시간에 GFP 생산을 따르도록 GFP 채널 모두에서 모든 50-10 분을 사용하여 사전 설정 위치의 수에 대한 이미지를 획득. </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>

저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

여기에 설명 된 방법에 몇 가지 단계가 매우 중요합니다. 우리는 여기서이 강조 표시됩니다. 밀도 기울기 원심 분리하여 호중구의 분리의 경우는 원심 분리 단계 도중 또는 이후 레이어를 방해하지 않는 것이 중요합니다. 플라스틱 피펫을 사용하여 호중구를 흡입 할 때, 세포의 층에서 동안으로 배출 액체 층을 방해 할 풍선을 짜내하지 있는지 확인하십시오. 또한, 시각적으?...

호중구 (PMNs)는 황색 포도상 구균 1에 타고난 면역 반응에 중요한 역할을 전문가 식세포이다. 호스트 및 미생물 사이의 지속적인 전투는 양쪽의 군비 경쟁을 주도하고있다. meticillin 내성 S.의 최근 사회 관련 (CA) 균주 구균 (MRSA)은 호중구 살해 2,3의 우회에 매우 효율적있을 것 등장했습니다. CA-MRSA의 과도한 페놀 용해 modulin (PSMS) 생산은 4,5 높은 독성과 관련되어있다. 인간 호중구 수용체 6 결합이 G-단백질의 활성화로 이어질 FPR2를 통해 이러한 PSMS를 인식 할 수 있습니다. 최초의 이벤트 중 하나는 칼슘의 세포 내 저장 (칼슘 2 +)의 동원이다. 칼슘 2 + 탈과립 및 식균 7 등 PMNs의 이펙터 기능의 다양한 보조 메신저 역할을합니다. 따라서 칼슘 2 +의 매우 중요한 지표이다PMNs를 활성화 PSMS의 기능 용량. 호중구에 PSMS의 효과를 연구하기 위해 신선한 호중구는 칼슘 동원에 형광하는 염료로 고립로드됩니다. 유동 세포 계측법 실험이 신속하게 동원은 측정 할 수 있습니다. 이 방법을 사용하면, 호중구에 대한 독성 및 기타 구성 요소의 직접적인 효과를 연구하고, 이러한 활성화 된 가장 낮은 농도를 측정 할 수 있습니다. 우리를 위해, 그것은 S.에 의해 만들어진 여러 단백질의 효과를 연구하기 위해 매우 유용한 도구입니다 구균은 FPR2 억제 단백질 (FLIPr) 8, 7 FLIPr 같은, 그리고 황색 포도상 구균 (CHIPS) 9 주 화성 억제 단백질로 면역 회피에 참여. 이러한 모든 단백질은 작용제를 인식 수용체에 결합하여 호중구의 칼슘 동원을 억제하기 위해 표시되었습니다. 최근 우리 그룹은 PSMS가 기능적으로 10 혈청 지질 단백질에 의해 억제되는 것을 설명하고있다 &lt;/&gt;을 먹다. 이 지단백은 PSMS 주로 세포 내 환경에서 그 기능을 발휘을 나타내는 혈액과 인체 조직 내에 다량으로 존재한다. 칼슘 동원 분석의 가용성은 우리가 정확하게 혈청의 매우 낮은 농도로 상당한 억제에 표시된 PSMS에 의한 호중구의 활성화에 혈청 지단백의 효과를 측정 할 수있었습니다. PSMS가 기능적으로 혈청에 의해 억제되기 때문에, 우리는 세포 내 독소 PSMS위한 중요한 기능이 있다는 가설을 세웠다. 따라서 우리는 식균 작용 후 PSMS의 역할을 결정하는 모색. 세포 사이 공간에 PSMS의 발현을 조사하기 위해, 우리는 GFP의 psmα 오페론의 프로모터의 기자 융합을 건설했다. 언제 S. 이러한 기자 변종 구균은 호중구에 의해 phagocytosed 세포했다, 표현의 유도는 형광 현미경 10을 사용하여 관찰 하였다. 물론이 절반hnique은 S.의 유전자의 다수의 표현의 연구를 수 식균 작용 후 구균이나 다른 병원균. S.에 대한 이후 간 틈새 시장에서 살아 남기 구균이 틈새 시장에서 활성화 유전자의 역할을 공부 타고난 면역 시스템 11 10 12를 극복하는 것이 매우 중요하다 그 독성을 이해하는 매우 관련이 있습니다.

<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

우리는 호중구에서 황색 포도상 구균에 의해 생산 분비 페놀 용해 modulins (PSMS) 및 기타 독소의 효과를 연구하는 방법을 제시한다. 우리는 밀도 기울기 원심 분리를 사용하여 신선한 호중구를 분리 호중구에 PSMS의 효과를 연구합니다. 이러한 호중구는 칼슘 동원에 형광 염료로로드됩니다. PSMS에 의한 호중구의 활성화는 무료로 세포 내 칼슘 농도의 신속하고 일시적인 증가를 시작합니다. 유동 세포 계측법 실험이 신속하게 동원은 무료 칼슘의 증가 농도 +에 반응하는 사전로드 된 염료의 형광을 모니터링하여 측정 할 수 있습니다. 이 방법을 사용 우리는 호중구를 활성화하고 호중구 활성화의 특정 및 일반 억제제의 효과를 측정하는 데 필요한 PSM 농도를 확인할 수 있습니다. 세포 내 공간 W에 PSMS의 발현을 조사하기전자 GFP에 PSMα 오페론의 프로모터의 기자 융합을 건설했다. 언제 S. 이러한 기자 변종 구균은 호중구에 의해 phagocytosed 세포 아르, 표현의 유도는 형광 현미경을 사용하여 관찰 할 수있다.

인간의 PMNs 칼슘 동원의 평가를위한 유세포 분석 FL-1 신호의 증가에 의해 표시되는 칼슘 플럭스에 의해 측정 급속한 활성화의 결과로 합성 PSMα3의 농도 일련의 호중구을 배양. 0.01 %의 합성 PSMα3의 사전 배양, 0.1 % 또는 1 % 혈청이 크게 칼슘 플럭스 (그림 1)을 유도 할 수있는 능력을 저해. 형광 현미경을 사용하여 PMNs하여 식균 작용 후 박테리아 GFP...

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

우리는에 의해 분비 PSMS 및 기타 독소의 효과를 연구하는 방법을 제시<em&gt; 황색 포도상 구균</em&gt; 유동 세포 계측법 및 형광 현미경을 사용하여 호중구합니다.

의 상호 작용을 연구<em&gt; 황색 포도상 구균</em&gt; 유동 세포 계측법 및 시간 경과 현미경에 의한 호중구와

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

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JoVE Journal

Immunology and Infection

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

13.0K 조회수.  University Medical Center Utrecht. 우리는에 의해 분비 PSMS 및 기타 독소의 효과를 연구하는 방법을 제시 황색 포도상 구균 유동 세포 계측법 및 형광 현미경을 사용하여 호중구합니다.

비디오: 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.

에 대한 드립 흐름 및 회전 디스크 원자로의 사용 포도상 구균 Biofilm 분석

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

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면역학 및 감염병학

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.

Methicillin 내성 황색 포도상 구균 아리 어스의 피하 감염 (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 ...

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

유동 세포 계측법에 의해 인간 호중구에 핵 팩터 활성화를 감지 할 간단하고 효율적인 방법

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.

유동 세포 계측법에 의해 주요 면역 세포 모집단과 형광 나노 입자의 상호 작용의 검출

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.

실시간으로 마취 마우스에 생체 내에 현미경으로 장간막 혈관에서 호중구와 단핵구 이미징

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.

의 유전자형<em&gt; 황색 포도상 구균</em&gt;에 의해 리보솜 스페이서 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.

수평 유전자 전달의 연구에 대한 방법론<em&gt; 황색 포도상 구균</em

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

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

절연, 특성화 및 비만 관련 염증에 의해 영향을 받는 조직에서 세포의 정화

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

고립과 Murine 인간과 치경 대 식 세포의 생체 외에서 문화

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

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.

마우스 대 식 세포에 의해 식 균 작용의 시간 경과 3D 영상

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

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