TB는 낭포 성 섬유증 캐나다에서 연구 교제를 인정합니다.

연구 윤리위원회 (REB)를 수집하고 인체에서 객담 샘플을 저장하는 데 필요합니다. 이 연구는 아픈 어린이 REB 번호 1000019444를 위해 병원에 의해 승인되었다. 1. 준비 CF 객담 샘플 <ol><li> 낭포 성 섬유증 클리닉에 일상적인 방문시 환자의 객담 샘플을 수집하고 얼음에 보관하십시오. </li><li> 처리를 받아야하는, 연구 실험실, 컬렉션의 첫 번째 시간 이내에 얼음에 객담 샘플을 전송. </li></ol> 2. 객담 처리 <ol><li> 얻어진 객담 시료의 양을 기록한다. 샘플 (즉, PBS 2 부 1 부 샘플)의 양을 2 배 인산염 완충 식염수 (PBS)를 추가한다. </li><li> 전송 피펫과 잘 샘플을 섞는다. 소용돌이 1 분 완전히 혼합하는 가장 높은 설정에 샘플. </li><li> 나누어지는 적절한 수의 1.5 ml의 마이크로 원심 튜브에 상기 혼합물 1 ㎖ 및 5,000 XG에 스핀 다운4 ℃에서 20 분. </li><li> 원심 분리 후, 상청액을 제거하고 펠렛을 버린다. </li><li> 필터 0.22 μm의 필터를 통해 뜨는을 소독하고 깨끗한 microcentrifuge 관에 수집합니다. 참고 : 여과 액의 무균는 LB 한천에 도금 및 액체 매체를 접종하여 시험한다. </li><li> 나중에 사용하기 위해 -80 ° C에서 가래 뜨는을 저장합니다. 참고 : 객담 여러 환자도 여과 다음 풀링 할 수 있습니다에서. </li><li> 객담 여과 액을 1/10 v / V를 원하는 미디어 (가래 100 ㎕, 미디어 900 μl를) 희석, 사용하기 전에. 참고 : 여기에, 표준 원성 국물 (LB) 미디어가 사용되었다. </li></ol> 바이오 필름 형성 3. 챔버의 coverglass 방법 <ol><li> (200 rpm)으로 진탕 37 ° C에서 원하는 미디어에 대한 관심이 하룻밤의 세균 분리를 성장. 주의 : 다른 박테리아의 수를 사용하여, 녹농균, 황색 포도상 구균이 임상 분리 포함부르크 홀데 리아의 cepacia의 복잡하고 아크 로모 박터의 아크 로모. 미디어의 선택은 그러나 LB 미디어가 초기 실험에 사용할 수있는 균주와 관심의 조건에 따라 달라집니다. </li><li> 밤새 배양 물로부터 신선한 미디어 4 ㎖ 중에 배양의 40 μl를 넣고 약 0.5 내지 600 nm의 (OD 600)에서의 광학 밀도를 갖는 배양을 얻었다 (200 RPM)를 흔들면서 37 ℃에서 3-4 시간 동안 성장 -0.6. </li><li> 10 % 객담 여과 또는 (제어 등) 객담 여과없이 원하는 미디어에서 1/5 단계 3.2에서 문화를 희석. 객담 여과 액의 다른 농도를 테스트 할 수 있습니다 (즉, 50 % 또는 100 %). </li><li> 슬라이드 챔버의 우물을 배정하는 것을 희석 200 μl를 사용합니다. </li><li> 박테리아가 흔들림없이 37 ° C에서 4 시간 동안 부착 할 수 있습니다. </li><li> 4 시간 후, 미디어를 제거하고 부드럽게 1 배 신선한 매체와 바이오 필름을 씻는다. 200 ㎕의 새로운 미디어로 교체하십시오. 참고 : 생물막의 가래를 Fres의 효과를 연구시간 미디어는 객담 뜨는을 포함해야합니다. </li><li> 바이오 필름은 현미경을위한 시간이 될 때까지 세척하지 않고, 미디어마다 12 시간을 대체 흔들림없이 37 ° C에서의 시간 원하는 시간 동안 성장하도록 허용합니다. 참고 : 생물막 항생제 감수성에 가래 상층 액의 효과를 연구하려면, 항생제는 생물막 성장의 24 시간을 다음과 같은 미디어에 추가되고 염색 및 바이오 필름의 영상까지 언론에 유지됩니다. </li></ol> 4. 염색 바이오 필름 및 공 초점 현미경 <ol><li> 원하는 성장 시간 (24 ~ 48 시간이 잘 작동)에 따라, 챔버 우물에서 미디어를 제거하고 부드럽게 멸균 PBS 300 μL로 두 번 각 챔버를 씻는다. </li><li> 필요한 솔루션의 각 ml의 대한 (생존 키트에 제공) 각 염료의 1 μl를 혼합하여 바이오 필름에 대한 염색 혼합물을 준비합니다. 물이나 미디어 솔루션에 염료를 확인합니다. 참고 : 물은 제조업체가 권장된다. </li><li> 챔버 covergla의 각 웰에 염료 혼합물 200 μl를 추가SS 45 분간 어둠 속에서 실온에서 부화. </li><li> 실에서 염색 혼합물을 제거하고 멸균 PBS 300 μL 잘 각을 씻는다. PBS를 제거하고 신선한 물 또는 용지로 교체합니다. </li><li> 공 초점 현미경을 통해 바이오 필름의 시각화를 진행합니다. </li></ol> 5. 공 초점 현미경으로 바이오 필름을 시각화 <ol><li> 즉시 (1 시간 이내) 염색 후 챔버 스테인드 생물막을 읽어보십시오. 한 번에 2 ~ 8 잘 챔버에 1을 염색하여 슬라이드의 시각화 지연을 최소화합니다. </li><li> 인수 여기 및 필터 세트의 레이저와 공 초점 현미경을 사용하여 이미지를 수행합니다. 참고 : 여기에, 스펙트럼 얼리스 레이저와 함께 회전하는 디스크 공 촛점 시스템 (녹색 : 491nm, 적색 : 561 nm의) 여기에 사용 하였다. 40분의 515 및 40분의 624의 배출 필터 세트는 생물막 생존 키트에서 얼룩을 시각화하는 데 사용되었다. </li><li> 카메라와 공 초점 현미경에 25X 물 목표를 사용하여 이미지를 가져 가라. </li><l난&gt; 사용 Z-스택은 바이오 필름을 모델링합니다. 모든 0.5 μm의는 생물막의 마지막에 초점 프레임에 첫 번째에 초점 비행기에서 시작하는 이미지를 가지고 (일반적으로 30 ~ 80 μm의 48 시간의 바이오 필름에 걸친) </li><li> 각 우물에서 3-5 이미지를 가져 가라. 참고 : 따라서 8 잘 챔버의 coverglass를 들어, 24-40 이미지가 생성됩니다. </li><li> 분석을 위해 이미지를 저장합니다. 참고 : OME-TIFF 파일이 COMSTAT (18, 19)를 사용하여 분석로서 이미지를 저장해야합니다. 생물막 이미지 분석에 대한 지침은 http://www.comstat.dk/에서 찾을 수 있습니다. 이미지가 수입되면, 이러한 각각의 채널 (빨강 및 녹색)의 평균 두께, 바이오 매스 및 표면 범위 등의 매개 변수를 분석 할 수 있습니다. </li></ol>

<ol>
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A., Dickson, A. C., Bruggeman, M. E., Bakaletz, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Biofilm+Formation+in+an+8-well+Chamber+Slide.">In vitro Biofilm Formation in an 8-well Chamber Slide.</a> J. Vis. Exp. (47), e2481 (2011).</li></ol>

본원에 기재된 방법은 외인성 제품의 존재 하에서 성장 세균 생물막의 시각화를 허용. 이러한 유형의 시스템을 사용하지 않을 때는 놀랍게도, exoproducts의 생산은 중요하다. 예를 들어, 디티 오 트레이 톨 (DTT)는 종종 샘플을 액화하기 위해 인간의 객담 샘플에 사용됩니다. 그러나, 단독 DTT의 효과 생물막 개발 가능성을 감소시킬 수있다 (데이터는 보이지 않음). 따라서, 모든 조건에 대한 적절한 통?...

세균 생물막은 서로에 부착되어 자기 분비 매트릭스 이탈되는 미생물의 기이다. 1,2- 고전, 그들은 물리적 흐름 조건 하에서 형성된 비 생물 적 또는 생물학적 표면에 부착 된 세균을 나타낸다. 바이오 필름은 또한 테스트 튜브에 형성된 열 풀 또는 펠리클의 공기 - 액체 계면의 표면과 같은 정적 상태 (흐름의 유무) 및 말단부에서 증가하는 것으로 나타났다. 그들은의 생물 연료, 부식 및 막힘의 결과, 저수지 또는 파이프에 형성 할 수있는 이러한 바이오 필름은 오래, 산업 공정에 큰 손해를 환경으로 인식하고 있습니다되었습니다. 3,4 그들은 카테터 관련된 감염, 낭포 성 섬유증 환자에서 폐 감염뿐만 아니라 수많은 다른 감염에 관여 된 바와 같이 바이오 필름은 또한 건강 관리 세팅에서 중요하다. 생물막 감염의 특징 중 5,6 하나는 드입니다항생제로 세균의 감수성을 주름과 선천성 면역 시스템에 의해 허가를 손상. 생물막 계 감염을 포함하는 7-9 가장 잘 연구, 임상 적 시나리오는 만성적 녹농균 생물막 감염된 낭포 성 섬유증 (CF), 환자에서 발생한다. 녹농균은 매우 어려운 치료를 할 수 있도록 만성 감염의 설립 동안 많은 변화를 겪을 수 있습니다. 10, 11 바이오 필름은 차등 선천성 면역을 활성화하고 염증을 구동 할 수있다. 이러한 감염은 CF 환자에서 증가 된 이환율 및 사망률을 초래할 12-14으로,이 컨텍스트에서 생물막 발달에 영향을 미칠 수있는 요인을 이해하는 것이 중요하다. 최근의 연구는 호스트 요인 녹농균 생물막 집계의 형성에 중요 제안합니다. 15이 바이오 필름은 항생제와 숙주 방어 메커니즘 감소 감수성에 기여한다. prese이러한 호중구 엘라 스타 제,뿐만 아니라, CF 폐에 존재하는 미생물로부터 분비 제품과 같은 숙주 유래 요인 NCE가 크게 생물막 형성 및 발달을 조절하는 잠재력을 가지고있다. (16) 또한, 바이오 필름은 다양한 경로의 발현을 조절하고 염증을 시작하는 호스트와 상호 작용합니다. 이러한 표준 크리스탈 바이올렛 분석 높은 처리량 방법, 생물막 공정에 관해서 몇 가지 정보를 제공 할 수 있지만, 이러한 요소에 대한 응답으로 바이오 필름의 시각화는보다 심층적 인 정보를 제공합니다. 이 논문에서는 체외 생물막의 개발 연구를 CF 환자의 객담에서 요소를 사용하는 방법을 설명한다. 이 방법은 상용 생물막 생존 키트를 사용 객담 함유 숙주 요인에 노출 생물막의 신속한 시각화 가능하다. 이 방법은 시각적 exogeno의 존재 생물막의 성장 중에 발생하는 변화를 식별하기 위해 사용될 수있다우리 제품 등 다양한 조건 하에서 생물막 개발의 변화를 분석하기위한 개선 된 방법을 나타낸다.

<table border="0" cellpadding="0" cellspacing="0" width="1270">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Lab-Tek II Chambered coverglass, #1.5 borosilicate, 8-well</td>
			<td style="width:159px;">Thermo Sicher Scientific</td>
			<td style="width:136px;">155409</td>
			<td style="width:729px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Filmtracer Live/Dead Biofilm Viabilty Kit</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">L10316</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Blood agar plates</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">R10215</td>
			<td>Confirming viability via CFU counts or selecting colonies for innoculation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">COMSTAT</td>
			<td style="width:159px;">Availble software online</td>
			<td style="width:136px;"></td>
			<td>COMSTAT is software to analyze biofilm images. Available www.comstat.dk&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Millers LB Broth</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>12780-052</td>
			<td style="width:729px;">Standard media for overnight gowth/biofilm growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Millex-GV Syringe Filters</td>
			<td style="width:159px;">Millipore</td>
			<td>SLGV013SL</td>
			<td>Filtering of sputum supernants</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Phosphate Buffered Saline (Dulbecco A)</td>
			<td style="width:159px;">Oxoid</td>
			<td style="width:136px;">BR0014G</td>
			<td>Washing of biofilm chambers after media removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss AxioVert 200M</td>
			<td style="width:159px;">Carl Zeiss</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hamamatsu C9100-13 EM-CCD</td>
			<td style="width:159px;">QS Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectral Borealis</td>
			<td style="width:159px;">Qs Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perkin Elmer Volocity</td>
			<td style="width:159px;">QS Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td>Instructions for this software can be found at: http://cellularimaging.perkinelmer.com/pdfs/manuals/VolocityuserGuide.pdf</td>
		</tr>
	</tbody>
</table>

visualizing the effects of sputum on biofilm development using a chambered coverglass model

바이오 필름은 자체 분비 매트릭스으로 쌌다 박테리아의 그룹으로 구성되어 있습니다. 이들은 산업 오염뿐만 아니라 많은 건강 관련 감염 현상 및 지속성에 중요한 역할을한다. 인간의 질병에서 가장 잘 설명하고 연구 바이오 필름 중 하나는 낭포 성 섬유증 환자의 만성 폐 감염으로 발생합니다. 호스트의 컨텍스트에서 생물막을 연구 할 때, 여러 가지 요인이 바이오 필름 형성 및 발달에 영향을 미칠 수있다. 호스트 요인 생물막 형성 및 발달에 영향을 미칠 수있는 방법을 확인하기 위해, 우리는 객담 상청액의 형태로 숙주 - 유도 인자의 존재 생물막의 성장에 고정 커브 글라스 챔버 법을 이용했다. 박테리아는 챔버에 접종 및 객담 여과 액에 노출되어있다. 성장의 48 시간 이후, 생물막 전에 공 초점 현미경 및 분석에 상업 생물막 생존 키트로 염색된다. 이미지 획득 후, 바이오 필름 특성은 다른 소프트웨어 플랫폼을 사용하여 평가 될 수있다.이 방법은 우리가 항생제를 포함한 다양한 물질의 존재 생물막 성장의 주요 특성을 시각화 할 수 있습니다.

실험의 전반적인 디자인은도 1에 표시된다. 이 프로토콜의 사용은 다양한 기간 (예, 24, 48 또는 72 시간) 동안 성장 생물막의 변화를 시각화하기위한 편리한 방법을 제공한다. 중요한 것은 이러한 객담 여액 같은 외인성 신호 생물막 개발의 변화를 시각적으로 첨가 할 수있다. 도 2에 도시 된 바와 같이, 10 % 객담 여액의 존재는 생물막의 구조 ...

Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

챔버의 coverglass 모델을 사용하여 바이오 필름 개발에 객담의 효과를 시각화

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

visualizing-effects-sputum-on-biofilm-development-using-chambered

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Immunology and Infection

8.3K 조회수.  Hospital for Sick Children. This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

비디오: Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Microtiter 디시 Biofilm 형성 분석

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

연구

면역학 및 감염병학

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

8 잘 회의소 슬라이드에서 체외의 Biofilm 형성에

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

유기 용매를 사용하지 않고 트리클로산의 효과 : RBL-2H3 비만 세포 탈과립에 화학 효과를 평가하는 마이크로 분석 실험

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

호스트 세포 RN​​A의 합성에 바이러스 감염의 효과를 측정하는 클릭 화학을 사용하여

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

를 사용하여 폐렴 구균 식민지에서 생균제의 효과를 조사<em&gt; 체외</em&gt; 준수의 분석

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

흐름에서 백혈구의 모집에 기질 세포의 영향 분석

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

PM10의 입자 바인딩 베팅 1 절 알레르기의 유세포 분석

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

실시간 이미징 및 2 단계 반복 흐름 시스템을 사용 하 여 곰 팡이 Biofilm 개발의 정량화

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are needed to decipher the utilization of microbiome modulators for human health benefits. However, the large-scale screening and optimization of modulators to validate microbiome modulation and predict related health benefits may be practically difficult due to the need for large number of animals and/or human subjects. To this end, in vitro or ex vivo models can facilitate preliminary screening of microbiome modulators. Herein, it is optimized and demonstrated an ex vivo fecal microbiota culture system that can be used for examining the effects of various interventions of gut microbiome modulators including probiotics, prebiotics and other food ingredients, aside from nutraceuticals and drugs, on the diversity and composition of the human gut microbiota. Inulin, one of the most widely studied prebiotic compounds and microbiome modulators, is used as an example here to examine its effect on the healthy fecal microbiota composition and its metabolic activities, such as fecal pH and the fecal levels of organic acids including lactate and short-chain fatty acids (SCFAs). The protocol may be useful for studies aimed at estimating the effects of different interventions of modulators on fecal microbiota profiles and at predicting their health impacts.

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

인체 배설물 미생물에 대한 중재 식이요법의 효과를 추정하는 시험관 내 배치 배양 모델

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn&#39;s-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn&#39;s-associated intestinal fibrosis.

In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn&#39;s fibrosis. We also discuss this approach in light of rapamycin facilitated inhibitory effects on intestinal fibrosis.

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has ...

챔버의 coverglass 모델을 사용하여 바이오 필름 개발에 객담의 효과를 시각화

요약

더 많은 비디오 탐색

이 비디오의 챕터

Microtiter 디시 Biofilm 형성 분석

8 잘 회의소 슬라이드에서 체외의 Biofilm 형성에

유기 용매를 사용하지 않고 트리클로산의 효과 : RBL-2H3 비만 세포 탈과립에 화학 효과를 평가하는 마이크로 분석 실험

호스트 세포 RNA의 합성에 바이러스 감염의 효과를 측정하는 클릭 화학을 사용하여

를 사용하여 폐렴 구균 식민지에서 생균제의 효과를 조사

흐름에서 백혈구의 모집에 기질 세포의 영향 분석

PM10의 입자 바인딩 베팅 1 절 알레르기의 유세포 분석

실시간 이미징 및 2 단계 반복 흐름 시스템을 사용 하 여 곰 팡이 Biofilm 개발의 정량화

인체 배설물 미생물에 대한 중재 식이요법의 효과를 추정하는 시험관 내 배치 배양 모델

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

챔버의 coverglass 모델을 사용하여 바이오 필름 개발에 객담의 효과를 시각화

요약

더 많은 비디오 탐색

이 비디오의 챕터

Microtiter 디시 Biofilm 형성 분석

8 잘 회의소 슬라이드에서 체외의 Biofilm 형성에

유기 용매를 사용하지 않고 트리클로산의 효과 : RBL-2H3 비만 세포 탈과립에 화학 효과를 평가하는 마이크로 분석 실험

호스트 세포 RN​​A의 합성에 바이러스 감염의 효과를 측정하는 클릭 화학을 사용하여

를 사용하여 폐렴 구균 식민지에서 생균제의 효과를 조사

흐름에서 백혈구의 모집에 기질 세포의 영향 분석

PM10의 입자 바인딩 베팅 1 절 알레르기의 유세포 분석

실시간 이미징 및 2 단계 반복 흐름 시스템을 사용 하 여 곰 팡이 Biofilm 개발의 정량화

인체 배설물 미생물에 대한 중재 식이요법의 효과를 추정하는 시험관 내 배치 배양 모델

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

호스트 세포 RNA의 합성에 바이러스 감염의 효과를 측정하는 클릭 화학을 사용하여