이 연구는 미국 농무부, 농무부 농업 연구소 (Agricultural Research Service)에 의해 지원되었다. 이 책에서 무역 이름 또는 상용 제품은 특정 정보를 제공 할 목적으로 만 언급하고 미국 농무부에 의해 추천이나 보증을 의미하지는 않습니다. USDA는 평등 한 기회 제공 및 고용주입니다.

1. 세균 콜로니의 주위 프린지 <ol><li> X를 성장 fastidiosa (Xf를) 테메 큘라 야생형 (22), pilG 삭제 돌연변이 PD2 매체 한천 플레이트 25에서 28 °에 (이전에 염색체 기반 유전자 보완 전략 (24) 기술 사용) (이전에 설명한 삭제 전략 (23)를 사용하여) Xf를 ΔpliG, 그 보완 XfΔpliG -C 5~7일에 대한 C. </li><li> 15 분 동안 121 ° C (249 ° F)에서 물에 오토 클레이브 셀로판 (1 × 1cm 2). , 셀로판의 한 조각을 들고 빈 페트리 접시에 셀로판의 한 구석을 터치하여 물기를 조심스럽게 한천 표면과 공기 건조의 15 % 이상 셀로판을 배치합니다. </li><li> 개별 X를 데리러 무균 한천 플레이트에 한천 표면의 15 %에 중첩 셀로판의 살균 장에 둥근 이쑤시개와 자리 세포 멸균와 fastidiosa 식민지. 접시를 품어2-3 일 동안은 28 ℃에서의. </li><li> 2 배 대물 렌즈와 해부 현미경 10 배 접안 렌즈를 사용하여 식민지의 가장자리의 형태를 검사합니다. 식민지 주변의 주변 프린지 사진. </li></ol> 2. 현미경과 미세 유체 흐름 챔버 <ol><li> 마찬가지의 포토 리소그래피 절차를 사용하여 제조하는 마이크로 유체 장치는 이전에 설명 5,18. 표준 리소그래피 방법을 이용하여 마스터 (26)의 실리콘 웨이퍼 상에 컴퓨터 보조 설계 소프트웨어와 4 개의 병렬 채널을 설계한다. </li><li> 폴리 디메틸 실록산 (PDMS)과 실리콘 웨이퍼 마스터에서 미세 유체 챔버를 만듭니다. 실리콘 웨이퍼 위에 마스터 비중 합 PDMS를 부어 1 시간 동안 60 ° C에서이를 경화. 웨이퍼 마스터에서 PDMS 복제를 벗겨과 유리 커버 슬립의 같은 크기로 22mm X 40mm로 블레이드와 PDMS 복제 트림. </li><li> PDMS의 복제, 유리 커버 슬립 (22 × 40 ㎜ × 2) 및 microsc 폭로2 분 27 30 W의 공기 플라즈마에 OPE 슬라이드 (51 X 76mm 2). 샌드위치 유리 커버 슬립과 마이크로 FL uidic 챔버를 구축 할 수있는 유리 현미경 사이의 PDMS의 몸. </li><li> 패터닝 된 채널의 각 단부에서 PDMS 관통 구멍 (5.5 mm 직경)을 뚫는다. 12-20cm 길이로 실리콘 고무 튜브를 잘라. PDMS의 복제본의 각 채널의 개방 단부에 실리콘 고무 튜브의 일단 (외경 5.1 mm, 내경 2.1 mm, 0.8 mm의 벽)를 넣고 1 시간 동안 60 ℃에서 비중 합 PDMS로 밀봉. </li><li> 플라스틱 루어 커넥터의 가시 끝에 튜브의 다른 쪽 끝을 연결합니다. 알루미늄 박으로 조립 된 미세 유체 챔버를 감싸고 20 분 동안이를 오토 클레이브. </li><li> X.의 균체를 모아서 스크래핑을 통해 fastidiosa 야생형, 돌연변이 Xf를 ΔpliG, 보완 XfΔpliG -C는 일회용 접종을 사용하여 PD2 매체 판에서 반복합니다. 세포 밀도를 조정PD2 브로스 600 nm에서 0.05의 광학 농도에 앞서 23 바와 같이. 1 ML의 기밀 주사기에 박테리아 세포 솔루션을 수집합니다. </li><li> 거꾸로 현미경의 무대에 미세 유체 장치를 탑재합니다. PD2 국물을 포함하는 5 ML의 기밀 주사기에 주입 관을 연결합니다. 주사기 펌프 5 ml의 기밀 주사기를 장착한다. </li><li> 폐기물 저장 용기에 배출 튜브를 연결합니다. 0.2 μL 분의 중간 유량을 유지, 30 분 -1 시스템을 안정화시키기 위해. </li><li> 박테리아 세포 솔루션을 포함하는 1 ml의 기밀 주사기에 사이드 입구 튜브를 연결합니다. 채널에 도달 할 때까지 고무 호스를 통해 박테리아 세포 용액을 플래시. 챔버의 사전 이미지 캡처에 언 바운드 세포를 세척하기 위해 0.2 μL 분의 매체 유량 -1 또 다른 30 분 동안을 유지한다. </li><li> 빛을 제어 할 현미경 분야 조정 부분 아래 현미경 셔터를 탑재. 셔터 공동으로 셔터를 연결ntrol 시스템 및 컴퓨터 셔터 제어 시스템을 연결한다. </li><li> 현미경의 비디오 포트에 디지털 카메라를 장착하고 컴퓨터에 연결합니다. 메뉴에서 &quot;셔터&quot;기능을 선택, 시간 경과 기록 작업 소프트웨어를 실행하고 소프트웨어와 셔터에 연결을 설정하기 위해 소프트웨어에서 자동으로 기본값으로 설치 셔터를 인식하고 있습니다. </li><li> 자동 소프트웨어 기본 촬상 장치로서 디지털 카메라를 인식 소프트웨어와 디지털 카메라와의 통신을 설정하는 저속 기록 소프트웨어 메뉴에서 &quot;디지털 카메라&quot;기능을 선택한다. </li><li> 20X 위상차 광학을 사용하여 채널들 중 하나에있는 박테리아 세포를 찾은 후, 촬상 전의 40X 대물 렌즈로 전환. </li><li> 현미경에서 이미지를 수집하기 위해 메뉴에서 기본 매개 변수를 사용하여 &quot;이미지 수집&quot;기능을 선택, 시간 경과 레코딩 소프트웨어를 실행합니다.다음으로, &quot;저속 획득&quot;기능을 열고 6-24 시간은 X. 연축의 운동성을 관찰하는 실험이 필요에 따라 지속 기간 30 초 5,18,28에 시간 간격을 설정 fastidiosa 5,18,28. 시간 경과 기록 작업을 시작합니다 &quot;OK&quot;를 클릭합니다. 선택 녹음을 마친 후 컴퓨터에서 대상 폴더에 이미지를 쌓아 &quot;다른 이름으로 저장&quot;메뉴에서 &quot;스택 기능&quot;을 클릭합니다. </li><li> 여러 채널의 경우, 6 시간 동안 첫 번째 채널에서 매 30 초 시간 경과 이미지를 캡처합니다. 표적 세포를 찾기 위해 다음 채널로 현미경의 대물 렌즈를 이동. 실험은 네 개의 채널을 이용하도록 설정되어있는 경우, 네 개의 채널이 순차적으로 각각의 이미지를 캡쳐하도록 전술 한 바와 같이 저속 기능을 반복한다. 한 세 가지로 일 연속 시간 경과 이미지 캡처를 계속합니다. 모든 실험은 실온 (23 ± 2 °의 C)에서 수행 하였다. </li><li> 에 시간 경과 이미지를 컴파일시간 경과 기록 영상 소프트웨어를 사용하여 비디오 파일. 실행 시간 경과 레코딩 소프트웨어, 메뉴에서 &quot;스택 기능&quot;을 클릭하고 컴퓨터에서 누적 된 파일을 열 &quot;오픈 스택 기능&quot;을 선택합니다. </li><li> 모든 이미지를 선택하고 &quot;AVI&quot;출력 형식을 선택, 스택 모듈에서 &quot;영화 만들기&quot;기능을 시작합니다. 컴퓨터의 대상 폴더에있는 동영상 파일을 저장하기 위해 &quot;다른 이름으로 저장&quot;을 클릭합니다. </li><li> 컴퓨터에 대상 폴더에서 컴파일 된 영화를 선택하고 재생합니다. 그리고, 상기 생성 된 영상 파일에 박테리아 세포의 운동성 경련 활성의 결과 시각화를 통해 단일 ​​세포의 운동성을 관찰한다. </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome-Based+Genetic+Complementation+System+for+Xylella+fastidiosa.">Chromosome-Based Genetic Complementation System for Xylella fastidiosa.</a> Appl. Environ. Microbiol. 75, 1679-1687 (2009).</li><li>Davis, M. J., Purcell, A. H., Thomson, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+Media+for+the+Pierce's+Disease+Bacterium.">Isolation Media for the Pierce's Disease Bacterium.</a> Phytopathology. 70, 425-429 (1980).</li><li>Xia, Y. N., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography.">Soft lithography.</a> Annu. Rev. Mater. Sci. 28, 153-184 (1998).</li><li>Chaudhury, M. K., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+interfacial+interactions+between+semispherical+lenses+and+flat+sheets+of+poly-(dimethylsiloxane)+and+their+chemical+derivatives.">Direct measurement of interfacial interactions between semispherical lenses and flat sheets of poly-(dimethylsiloxane) and their chemical derivatives.</a> Langmuir. 7, 1013-1025 (1991).</li><li>Cruz, L. F., Parker, J. K., Cobine, P. A., De La Fuente, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-enhanced+twitching+motility+in+Xylella+fastidiosa+is+linked+to+a+single+PilY1+homolog.">Calcium-enhanced twitching motility in Xylella fastidiosa is linked to a single PilY1 homolog.</a> Appl. Environ. Microbiol. 80, 7176-7196 (2014).</li></ol>

저자는 더 경쟁 재정적 이해 관계가 없음을 선언합니다.

본 연구에서는 X의 모션 동작을 특징으로하는 fastidiosa PilG 돌연변이 Xf를 ΔpilG와 새롭게 디자인 된 다수의 병렬 나노 채널 마이크로 FL uidic 챔버에서의 보완 XfΔpilG- C 균주. 새롭게 디자인 된 마이크로 FL uidic 챔버는 하나의 50 μm의 넓은 채널 (18)과 이전 디자인에 비해 폭이 100 μm의 나노 채널과 최대 4 개의 병렬 챔버를 가질 수 있습니다. 개선 된 넓?...

Xylella의 fastidiosa가 포도에서 피어스 질환 (비 티스 VINIFERA L.) 1, 2, 3을 포함하여 경제적으로 중요한 작물 질환들을 유발하는 그람 - 음성 비 flagellated 병원성 박테리아이다. 이는 박테리아 물 전도성 목부에 한한다 선박. 소문의 감염은 목부의 선박과 물 부족의 결과와 영양 결핍 (3)의 막힘이 발생합니다. 성공적인 정착 식물 (3)의 나머지 감염의 초기 위치에서 이동하는 박테리아의 능력에 달려있다. X. 특징으로 한 극성 타입 IV의 필리 (4)의 연장, 첨부 한 후퇴 통해 편모 독립적 박테리아 이동 수단이되어 운동성 경련 fastidiosa 5,6,7. 경련 운동성 레이저 핀셋 및 원자 힘 현미경 (AFM) 8,9,10에 의해 관찰되었다. 이러한 기술을 이용하여 t매혹적인의 motilities는 N.의 유형 IV의 pilus에 의해 생성 임질과 P. 녹농균은 플로리다 uorescently 라벨 필리 현미경으로 그들의 움직임을 포착 특징으로했다. 두 방법은 개별 박테리아의 접착력 상술되었지만, 절차는 복잡하고 시간 소모적 9,10이다. 마이크로 FL uidic 챔버 장거리 개별 셀의 이동뿐만 아니라 박테리아 세포 5,6- 작은 응집체를 관찰 하였다. 이 챔버는 시간 경과 영상 녹화 시스템 11,12,13,14 통합 접시에 미세 나노 채널로 설계되었다. 마이크로 uidic 챔버 장치 박테리아의 이동 동작 및 세포 - 세포 상호 작용을 연구 몇 가지 장점을 제공 FL : (ⅰ)는 다중 채널 기능이 통합 된 플랫폼을 제공한다; (ⅱ)는 모션 및 박테리아의 나노 크기 특성의 단셀의 집합체를 검사 할 수있다; (ⅲ)이 직접 m 가능박테리아 세포 및 시간 경과 분석 icroscopic 영상 기록은, (ⅳ)는 마이크로 환경에서 박테리아의 개인 및 / 또는 인구의 장기 공간 및 시간 관찰을 제공한다 (V) 채널에서 배양액의 유량을 정확하게 제어 할 수 있으며, 배양액 (VI)의 매우 작은 양 (1 ml)을 각각의 실험에 대해 요구된다. 최근 마이크로 FL uidic FL 흐름 시스템은 다양한 미세 환경 14,15,16에서 박테리아 세포의 행동을 조사하기 위해 사용되어왔다. 접착 및 E.의 표면에 부착 대장균 (15), X. fastidiosa 16 및 Acidovorax 유리 표면에 14 마이크로 FL uidic 챔버를 이용하여 평가 하였다 citrulli. Acidovorax의 citrulli의 유형 IV의 필리에 의해 매개 집계 및 바이오 Fi를 필름 형성 (14)를 분석 하였다. A.의 또한, 움직임 citrulli는 플로리다 흐름 C 관찰onditions는 타입 IV의 필리가 식민지에서 중요한 역할을 할 수 있음을 입증하고 A.의 확산 수액 FL 흐름 조건에서 목부 선박에 citrulli. 녹농균 및 X의 꿈틀 motilities fastidiosa 세포가 성공적으로 미세 유동 챔버 5,6,17에 유체 전류에 대해 관찰되었다. 타입 IV pilus는 X의 pilB 및 pilQ 돌연변이 결핍 fastidiosa는 깊이 마이크로 FL uidic 장치 5,6,18에서 플로리다 유동 조건에서 운동성을 꿈틀의 속도를 변경하는 것으로 밝혀졌다. 마이크로 FL uidic 장치 세균 부착 및 운동성 실시 연구 마이크로 FL uidic 챔버들은 시험 관내에서 필리 매개 세균의 경련 운동성 및 이동 분석에 특히 적합한 것으로 나타났다. 이러한 결과는 세포 - 세포 부착, 응집 집락 내를 용이 경련 매개 이동기구를 설명호스트는 결국 전신 감염으로 이어질. X의 필 - 열병합 오페론 fastidiosa는 인코딩 신호 전달 (20) 경로 pilG, PILI, pilJ, 알약, chpB 및 chpC이 포함되어 있습니다. 횡단 chemoreceptors는 주변 세포질 도메인에 화학적 자극을 결합하여 궁극적으로 세균 꿈틀 운동성을 제어하기 위해 자신의 세포질 부분에 신호 캐스케이드를 활성화합니다. X의 필 - 열병합 오페론에서 fastidiosa하는 포스 포 셔틀 단백질 PilG는 최 회장에 동족체이다. E.에서 대장균 및 P. 녹농균은 최은 편모 단백질 모터 (19), (21)와 상호 작용 화성 시스템에서 응답 레귤레이터이다. X의 독성을 향해 필 - 열병합 오페론의 기여 있지만 fastidiosa는 환경 신호에 응답하고 X의 규제 / 모터 유형 IV의 필리에, 최근에 화성 오페론에 pilG의 역할을 (20)을 조사 하였다 fastidiosa는 UNC입니다리어. X의 꿈틀 운동의 활동에 화성 레귤레이터 pilG의 통찰력을 규명하려면 fastidiosa, 마이크로 FL uidic 챔버는 X의 꿈틀 운동성을 평가하는 데 사용됩니다 fastidiosa. X.의 fastidiosa의 pilG은 삭제 돌연변이 Xf를 ΔpliG, 보완 변형 XfΔpliG -C 및 시험 관내에서의 야생형의 표현형을 비교하는 것을 특징으로한다. 결과는 X의 경련 운동성 pilG의 역할을 강조 fastidiosa.

<table>
	<tbody>
		<tr>
			<td>Biology materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X. fastidiosa (Xf) Temecula wild type</td>
			<td></td>
			<td></td>
			<td>Costa, H. S., et al., 2004 22</td>
		</tr>
		<tr>
			<td>pilG deletion mutant Xf&#916;pliG</td>
			<td></td>
			<td></td>
			<td>Shi, X. Y., et al., 2007 26</td>
		</tr>
		<tr>
			<td>pilG complementary strain Xf&#916;pliG-C&#160;</td>
			<td></td>
			<td></td>
			<td>Davis, M. J., wt al. 1998 23</td>
		</tr>
		<tr>
			<td>Physical materials and equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable inoculating loops</td>
			<td>VWR international, Radnor, PA</td>
			<td>#22-363-607</td>
			<td>quantitative procedures such as bacterial collection</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning Corporation</td>
			<td>#0002709226</td>
			<td>Sylgard 184 silicone Elastomeric Kits</td>
		</tr>
		<tr>
			<td>AmScope MD2000 digital camera</td>
			<td>AmScope, Irvine, CA</td>
			<td>SE305R-AZ-E</td>
			<td>Image, video recording and measurement&#160;</td>
		</tr>
		<tr>
			<td>Tubes line</td>
			<td>Edgewood, NY</td>
			<td>#T4300</td>
			<td>Connected to the syringe and microfluidic chamber</td>
		</tr>
		<tr>
			<td>Plastic luer connectors</td>
			<td>Edgewood, NY</td>
			<td></td>
			<td>Connected to the syringe and microfluidic chamber</td>
		</tr>
		<tr>
			<td>Syringe pumps</td>
			<td>Pico Plus, Harvard Apparatus, MA</td>
			<td>#702209</td>
			<td>The flow rate can be adjusted while the pump is running.</td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>Gastight, Hemilton Company, Reno, NV</td>
			<td>#1005</td>
			<td>Provide the flowing broth</td>
		</tr>
		<tr>
			<td>Inverted Olympus IMT-2 microscope</td>
			<td>Olympus</td>
			<td>IMT-2 FLuoro PHase</td>
			<td>Image observation and recording</td>
		</tr>
		<tr>
			<td>SPOT-RT digital camera</td>
			<td>Diagnostic Instruments, Inc., MI</td>
			<td>RT230</td>
			<td>Image, video recording and measurement</td>
		</tr>
		<tr>
			<td>Microscope Shutter</td>
			<td>The UNIBLITZ, US</td>
			<td>#LS2T2</td>
			<td>Control camera&#8217;s exposure time</td>
		</tr>
		<tr>
			<td>Microscope Shutter Control system</td>
			<td>The UNIBLITZ, US</td>
			<td>VCM-D1</td>
			<td>VCM-D1 Single Channel CE/UL/CSA Approved Shutter Driver</td>
		</tr>
		<tr>
			<td>MetaMorph Image software</td>
			<td>Universal Imaging Corp., PA</td>
			<td></td>
			<td>Real-time super-resolution image processing&#160;</td>
		</tr>
	</tbody>
</table>

visualization of twitching motility and characterization of the role of the pilg in xylella fastidiosa

Xylella fastidiosa의 식물의 경제적으로 중요한 질환들을 유발하는 그람 - 음성 박테리아 이외 flagellated이다. 꿈틀 운동성은 X를 제공합니다 fastidiosa 장거리 내 공장 이동 및 정착을위한 수단이 X에서 병원성으로 기여 X의 fastidiosa. 꿈틀 운동 fastidiosa는 타입 IV의 필리 운영하고 있습니다. Xylella의 fastidiosa의 유형 IV의 필리는 pilG, 신호 전달 경로에 관여하는 필-순찰대 오페론 인코딩 단백질의 화성 조절기에 의해 조절된다. X의 꿈틀 운동에 pilG의 역할을 규명하려면 fastidiosa하는 pilG 결핍 돌연변이 Xf를 ΔpilG 및 기본 pilG을 포함하는 그것의 보완 변형 XfΔpilG- C가 개발되었다. 타임 랩스 영상 녹화 시스템과 통합 마이크로 유체 챔버 XfΔp 경련 운동성을 관찰하는 데 사용ILG, XfΔpilG- C와 야생형 균주. 이 기록 장치를 사용하여, 장기 시공간 응집 관측 각 셀 및 경련 운동성 통해 박테리아 집단의 이동을 허용한다. X. 돌연변이 XfΔpliG는 연축 결핍 표현형을 나타내 fastidiosa 반면 야생형과 상보 XfΔpilG- C 균주 직접 미세 유동 챔버 관찰 전형적인 경련 운동성 특성을 보였다. 이 연구는 pilG는 X의 꿈틀 운동성에 기여하고 있음을 보여줍니다 fastidiosa. 마이크로 유체 유동 챔버 경련 운동성을 관찰하는 수단으로 사용된다.

IV 형 pilus 매개 경련 운동성을 나타내는 콜로니 주변 무늬의 존재는, X의 콜로니가 관찰되었다 fastidiosa 야생형 및 보완 Xf를 ΔpliG -C 변형 (그림 1). 돌연변이 XfΔpliG 그러나, 콜로니 주변부 프린지를 (도 1)을 나타내지 않았다. 나노 미세 유체 흐름 챔버에 박테리아 세포의 시간 경과 영상은 꿈틀 운동이 모두 야생형 <...

Watch this Scientific Journal Video about Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa at JoVE.com

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

본 연구에서는 나노 미세 유체 흐름 챔버는 시각화 기능 Xylella fastidiosa, 포도 나무에 피어스의 질병을 일으키는 세균의 꿈틀 운동의 특성을 사용 하였다.

의 역할의 꿈틀 운동성의 시각화 및 특성<em&gt; PilG</em&gt;에서<em&gt; Xylella의 fastidiosa</em

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility ...

The over goal of this protocol is to functionally characterize the genes responsbile for the twitching motility of bacteria using a nano microfluidic flow chamber integrated with a time-lapse image recording system.This method can help answer key questions about a bacteria phenotype and associated gene function, such as the twitching motility of bacteria.The main advantage of this technique is that it provides a special and a tempered observation for aggregation migration of individual cells and populations.Demonstrating this procedure will be Dr.Xiangyang Shi, a post-doc from my laboratory.Using a standardized deletion strategy, grow Temecula wild-type Xylella fastidiosa, grow pilG deletion mutants, and grow the complimentary strain all on PD2 medium agar plates.Incubate the bacteria at 28

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Immunology and Infection

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

10.6K 조회수.  University of California, Davis. The over goal of this protocol is to functionally characterize the genes responsbile for the twitching motility of bacteria using a nano microfluidic flow chamber integrated with a time-lapse image recording system.This method can help answer key questions about a bacteria phenotype and associated gene function, such as the twitching motility of bacteria.The main advantage of this technique is that it provides a special and a tempered observation for aggregation migration of individual cells ...

비디오: Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

백신에 마우스 응답 Nasopharyngeal - 관련 Lymphoreticular 조직 (NALT)의 역할을 검토

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

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면역학 및 감염병학

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Phagokinetic 트랙 운동성 분석에 의한 세포 마이그레이션의 수량 평가

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

정성 및 정량 검출을위한 시험 법 및 단백질 항생제 특성

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

혈액 처리를위한 빠른 방법은 인간의 혈액에서 혈장 펩타이드 수준의 수율을 증가하는

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

세균성 접착 및 혈 청 저항에 Vitronectin의 역할을 공부에 대 한 분석

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

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 Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) containing immunologically active molecules. Knowledge regarding the molecular mechanisms of vesicle biogenesis, the content of the vesicles, and their functions at the pathogen-host interface is very limited. Addressing these questions requires rigorous procedures for isolation, purification, and validation of EVs. Previously, vesicle production was found to be enhanced when M. tuberculosis was exposed to iron restriction, a condition encountered by Mtb in the host environment. Presented here is a complete and detailed protocol to isolate and purify EVs from iron-deficient mycobacteria. Quantitative and qualitative methods are applied to validate purified EVs.

Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

철분 제한 마이코박테리아에 의해 생성된 세포외 소포의 격리 및 특성화

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) ...

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

종양 미세 환경에서 면역 세포 집단의 시각화, 정량화 및 매핑

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

형광 항생제 프로브를 사용한 세균 저항성 시각화

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be altered by severe brain injury. This is evidenced by the fact that pneumonia is a major cause of mortality in patients who have suffered from ischemic stroke. The goal of this protocol is to describe the use of multicolor flow cytometric analysis to identify 13 types of immune cells in the lungs of mice, including alveolar macrophages, interstitial macrophages, CD103+ or CD11b+ dendritic cells (DCs), plasmacytoid DCs, eosinophils, monocytes/monocyte-derived cells, neutrophils, lymphoid-derived&#160; T and B cells, NK cells, and NKT cells, following ischemic stroke induction by transient middle cerebral artery occlusion. Moreover, we describe the preparation of lung homogenates using a bead homogenization method, to determine the expression levels of 13 different cytokines or chemokines simultaneously by multiplex bead arrays coupled with flow cytometric analysis. This protocol can also be used to investigate the pulmonary immune response in other disease settings, such as infectious lung disease or allergic disease.

This protocol describes the use of flow cytometry to identify the changes in immune cell composition, cytokine profile, and chemokine profile in the pulmonary environment following transient middle cerebral artery occlusion, a murine model of ischemic stroke.

폐 환경에서 면역 세포 및 전염증성 중재자의 특성화

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be ...