Name: conjunctival commensal isolation and identification in mice
Uploaded: 2021-05-01T13:00:00
Description: Watch this Scientific Journal Video about Conjunctival Commensal Isolation and Identification in Mice at JoVE.com

P30 DK034854의 자금 지원VY, LB 및 매사추세츠 호스트-미생물군유전체 센터의 연구 및 NIH/NEI R01 EY022054의 자금 지원 MG.

마우스와 관련된 모든 절차는 기관 동물 관리 및 사용위원회 지침을 따릅니다. 미생물 및 잠재적으로 오염된 물질로 작업할 때 실험실 안전 지침(기관 환경 보건 및 안전 부서의 지시)을 따르십시오. 잠재적으로 생물학적 오염 물질을 폐기하기 전에 적절한 폐기물 리셉터클 및 오염 제거 절차를 사용합니다.
1. 눈 면봉 준비, 작업 장 설정, 마우스 눈 면봉 및 샘플 농축
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안구 표면의 파시균 상태로 인해 많은 실험실에서 안구 공생을 분리하는 데어려움이 있어7,20의성장, 낮은 풍부및 낮은 다양성8을가진 시료수가 적어집니다. 이 방법은 MALDI-TOF MS에 의한 재설계된 눈 면봉 및 식별뿐만 아니라 농축 단계의 첨가에 의해 표준 배양 관행4,21에 크게 향상됩니다. ?...

이 프로토콜의 목적은 안구 내막 에서 실행 가능하고 희귀 한 호기성 및 학부 성 혐기성 미생물의 특정 절연을 강화하여 안구 미생물군유전체를 특성화하는 것입니다. 광범위한 연구는 피부, 창자, 호흡기 및 생식기에 대한 막수 점막 커뮤니티를 프로파일드하고 이러한 지역 사회가 면역 체계의 발달과 반응1,2,3에영향을 미친다는 것을 보여줍니다. 안구 내문 공동체는 안구건조증4,쇼그렌 증후군5 및 당뇨병6과같은 특정 질환 병리학 중에 변화하는 것으로 나타났다. 그러나, 전형적인 안구 표면 발생 공동체를 정의하는 능력은 다른 점막부위에비해 상대적으로 낮은 풍요로움에 의해 방해된다6,7,8. 이것은 상주 안구 미생물군유전체가 있는지, 그리고 존재하는 경우에, 피부 microbiome와 다른지, 결과적으로, 타고난 면역 계통 발달 및 반응에 그것의 현지 효력이 있는지를 통해 논쟁을 자극합니다. 이 프로토콜은 이 질문을 해결하는 데 도움이 될 수 있습니다.
일반적으로, 안구 공생 틈새 시장을 정의하는 접근법은 시퀀싱 및 문화 기반 기술4,7,9에기초한다. 16 S rDNA 염기서열 분석 및 BRISK 분석7은 배양 기반 기술보다 더 넓은 다양성을 나타내지만 살아있는 미생물과 죽은 미생물을 구별할 수는 없습니다. 안구 표면은 눈물 필름의 항균 특성4로 인해 많은 미생물에 적대적이기 때문에 많은 DNA 단편을 생성하며, DNA 기반 접근법은 오염물질이 아닌 상주 교향곡으로 죽은 박테리아의 식별으로 데이터를 왜곡할 수 있는 이러한 유물을 검출할 것이다. 이로 인해 미생물의 풍부와다양성(10)에서더 높은 것으로 안구 공간의 비정상적인 장신 식별 및 특성화가 발생한다. 이것은 DNA 기지를 둔 방법을 통해 상주 안구 미생물군유전체를 정의하는 것을 어렵게 합니다. 반면, 표준 배양 기반 기술은 하중이 너무 낮기 때문에11을감지할 수 없습니다. 우리의 방법은 결막을 표적으로 할 수 있는 얇은 면봉을 사용하여 표준 관행을 향상시키고, 따라서 이웃 피부에서 오염을 피하고, 실행 가능한 유기체가 가능한 그러나 비 culturable를 소생하는 것을 목표로 영양 조밀한 매체에 있는 짧은 문화에 의해 풍부해질 수 있다는 개념뿐만 아니라, 희귀 한 생존 가능한 미생물을 위해 풍부하게 합니다.
결과, 눈 면봉 당 안구 공자의 상대적인 풍부, 결막 상주 미생물군유전체를 특성화 하 고 비교 목적을 위해 중요 하다. 우리의 데이터는 피부와 결막 미생물의 차이뿐만 아니라 증가 된 나이와 풍부한 성별 특정 차이와 더 큰 다양성이 있음을 보여줍니다. 더욱이, 이러한 접근법은 녹아웃마우스(12)에서의기생 차이를 재현적으로 발견했다. 이 프로토콜은 caging 관행, 지리, 또는 질병 상태뿐만 아니라 면역 계통 발달 및 반응에 대한 교만 대사 산물 및 제품의 국소 효과로 인해 달라질 수 있는 안구 미생물군유전체를 설명하기 위해 적용될 수 있다.

<table>
	<tbody>
		<tr>
			<td>0.1 to 10 &#181;l pipet tip</td>
			<td>USA Scientific</td>
			<td>1110-300</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>0.5 to 10 &#181;l Eppendorf pipet</td>
			<td>Fisher Scientific</td>
			<td>13-690-026</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Fisher Scientific</td>
			<td>BD309623</td>
			<td>1 syringe for each eye swab group</td>
		</tr>
		<tr>
			<td>1.5 ml Eppendorf tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>1000 &#181; ml pipet tip</td>
			<td>USA Scientific</td>
			<td>1111-2021</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>200 to 1000&#181;l Gilson pipetman (P1000)</td>
			<td>Fisher Scientific</td>
			<td>F123602G</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G needle</td>
			<td>Fisher Scientific</td>
			<td>14-826AA</td>
			<td>1 needle per eye swab group</td>
		</tr>
		<tr>
			<td>3 % Hydrogen Peroxide</td>
			<td>Fisher Scientific</td>
			<td>S25359</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176; C Incubator</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70 % Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>PX1840-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ana-Sed Injection (Xylazine 100 mg/ml)</td>
			<td>Santa Cruz Animal Health</td>
			<td>SC-362949Rx</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BBL Gram Stain kit</td>
			<td>Fisher Scientific</td>
			<td>B12539</td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen Burner</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clean paper towels</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Batting/Sterile rolled cotton</td>
			<td>CVS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 1 ml Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-711-9AM</td>
			<td>for Gram stain and catalase tests</td>
		</tr>
		<tr>
			<td>E.coli</td>
			<td>ATTC</td>
			<td>ATCC 8739</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-A3</td>
			<td>for Gram stain and catalase tests</td>
		</tr>
		<tr>
			<td>Ketamine (100mg/ml)</td>
			<td>Henry Schein</td>
			<td>9950001</td>
			<td></td>
		</tr>
		<tr>
			<td>Mac Conkey Agar Plates</td>
			<td>Fisher Scientific</td>
			<td>4321270</td>
			<td>store at 4 &#176;C until ready to use</td>
		</tr>
		<tr>
			<td>Mannitol Salt Agar</td>
			<td>Carolina Biological Supply</td>
			<td>784641</td>
			<td>Prepare plates according to mfr&#39;s instructions, store at 4 &#176;C for 1 week</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Jackson Labs</td>
			<td>C57/BL6J</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-12</td>
			<td>for Mannitol Salt agar plates</td>
		</tr>
		<tr>
			<td>RPI Brain Heart Infusion Media</td>
			<td>Fisher Scientific</td>
			<td>50-488525</td>
			<td>prepare according to directions and autoclave</td>
		</tr>
		<tr>
			<td>SteriFlip (0.22 &#181;m pore size polyester sulfone)</td>
			<td>EMD/Millipore, Fisher Scientifc</td>
			<td>SCGP00525</td>
			<td>to sterilize anesthesia</td>
		</tr>
		<tr>
			<td>Sterile Corning Centrifuge Tube</td>
			<td>Fisher Scientific</td>
			<td>430829</td>
			<td>anesthesia preparation</td>
		</tr>
		<tr>
			<td>Sterile mouse cage</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tooth picks (round bamboo)</td>
			<td>Kitchen Essentials</td>
			<td></td>
			<td>autoclave before use and swab preparation</td>
		</tr>
		<tr>
			<td>Trypticase Soy Agar II with 5% Sheep&#39;s Blood Plates</td>
			<td>Fisher Scientific</td>
			<td>4321261</td>
			<td>store at 4 &#176;C until ready to use</td>
		</tr>
		<tr>
			<td>Vitek target slide</td>
			<td>BioMerieux Inc. Durham,NC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vitek-MS</td>
			<td>BioMerieux Inc. Durham,NC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vitek-MS CHCA matrix solution</td>
			<td>BioMerieux Inc. Durham, NC</td>
			<td>411071</td>
			<td></td>
		</tr>
		<tr>
			<td>Single use eye drops</td>
			<td>CVS Pharmacy</td>
			<td></td>
			<td>Bausch and Lomb Soothe Lubricant Eye Drops, 28 vials, 0.02 fl oz. each</td>
		</tr>
	</tbody>
</table>

conjunctival commensal isolation and identification in mice

안구 표면은 한 번 면역 특권과 비생물으로 간주되었다, 하지만 최근에는 작지만 지속적인 막연한 막대한 존재가 있는 것으로 보인다. 안구 점막에서 세균 종의 식별 및 모니터링은 그들의 낮은 풍부와 인문 성장 및 식별을위한 적절한 방법론의 제한된 가용성으로 인해 도전하고있다. 문화 기반 또는 DNA 염기서열 분석 방법의 두 가지 표준 접근 법이 있습니다. 첫 번째 방법은 제한된 회수 가능한 박테리아로 인해 문제가 되고 두 번째 접근법은 안구 공간의 비정상적인 표현으로 이어지는 살아있는 박테리아와 죽은 박테리아를 모두 식별합니다. 우리는 표준 미생물 배양 기술을 기반으로 세균 격리를 위한 견고하고 민감한 방법을 개발했습니다. 이것은 "실험실"이 낮은 결막을 대상으로 하는 얇은 면봉을 활용한 면봉 기반 기술이며, 그 다음으로 호기성 및 학부 해부학 적 제네라에 대한 증폭 단계가 뒤따릅니다. 이 프로토콜은 우리가 코린 박테리움 spp., Coagulase 네거티브 황색포도상구균 spp., 연쇄상 구균 spp등과 같은 결막 종을 분리하고 식별할 수 있게 했습니다. 접근은 다른 질병 조건의 마우스에 있는 막증을 정의하는 것이 적합합니다.

도금에 대한 다양한 방법을 보여주는 눈 면봉 판에 대한 대표적인 결과는 C57BL/6 마우스로부터 형태학적으로 다양한 분리를 보여주는 도 3A에 그려지고 있다. 각 뚜렷한 격리에 대해, 식민지는 스트립과 상대적 풍부, 눈 면봉 당 독특한 콜로니 형성 단위 (CfU)에 계산하고 비교 목적을 위해 플롯하였다. 미생물 특성화를 위해, 박테리아는 마스터 TSA 플레이트를 생산하기 위해 개...

Watch this Scientific Journal Video about Conjunctival Commensal Isolation and Identification in Mice at JoVE.com

Conjunctival Commensal Isolation and Identification in Mice

여기에 제시된 것은 미생물 기반 방법 및 MALDI-TOF 질량 분석법에 의한 후속 식별을 통해 독특한 눈 면봉 및 배양 기반 농축 단계를 사용하여 호기성 및 학부성 혐기성 마우스 결막 성 세균의 격리 및 증폭을 위한 프로토콜이다.

마우스의 결막 장금 격리 및 식별

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. ...

이 방법은 눈에 적대적인 환경 때문에 도전적인 가능한 안구 결막 미생물을 식별합니다. 안구 미생물군유전체가 존재하는지, 안구 질환의 세균성 존재가 어떻게 변하는지에 대한 답을 하는 데 도움이 될 수 있습니다. 실행 가능한 미생물과 비실행성 미생물을 모두 식별하는 DNA 염기서열분석과는 달리,이 방법은 가능한 미생물만 식별하여 안구 공생 커뮤니티에 대한 명확한 이해를 합니다.연구에 따르면 비자가면역및자가면역안구건조증과 같은 안구 질환을 진단하기 어려운 것은 독특한 안구 세균성 존재가 있는 것으로 내다볼 수 있다. 이 방법은 특유의 미생물 서명으로 안과 질환을 진단하는 데 사용될 수 있습니다. 먼저 면봉할 마우스 수에 적합한 양의 면 타격과 이쑤시개를 자동화한 다음, 면 타격의 1/2 센티미터 길이조각을 꼬집고 가장자리를 당겨 서 타격을 막기 직전에 멈추는 평평한 단일 다공성 층을 형성하여 애타게 합니다.이쑤시개 끝에 늘어진 조각을 가볍게 잡고 이쑤시개 끝의 날카로운 끝 중 하나 주위에 타격을 소용돌이치십시오. 완성 된 눈 면봉은 팁에서 약 1/2 ~ 1 센티미터 까지 뻗어 있는 팁 위에 뻗어있는 면의 매우 얇은 층을 갖게됩니다. 면봉을 작은 비커에 넣고 옆으로 면봉한 다음 덮고 오토클레이브합니다.오염을 최소화하기 위해 소독제로 작업 영역을 청소하고 멸균 뇌 심장 주입의 알리쿼트 0.5 밀리리터, 또는 BHI 미디어를 라벨1.5 밀리리터 멸균 마이크로 원심분리기 튜브로 분류합니다. 랙에 튜브를 캡하고 얼음에 랙을 설정합니다. 실험 마우스, 빈 멸균 케이지, 실온 마취, 25 게이지 바늘 및 1 밀리리터 주사기가있는 케이지를 포함하는 마우스 마취 스테이션으로 시작하여 왼쪽에서 오른쪽으로 워크플로우를 설정합니다.다음으로, 얼음에 알리인용 BHI, 멸균 된 눈 면봉, 윤활 점안액, 생체 위험 용기, 깨끗한 종이 타월 및 70 %의 이소프로판올 스프레이가 들어있는 눈 면봉 스테이션을 준비하십시오. 마지막으로 실온 혈액 화천 플레이트, 10 마이크로리터 파이펫, 10 마이크로리터 멸균 일회용 팁 및 생체 위험 폐기물 용기가있는 도금 스테이션을 설치합니다. 뒷발 패드를 짜서 마우스가 제대로 마취되었는지 확인하십시오.움직임이 없는 경우에만 진행합니다. 마취 된 마우스와 다른 손을 처리하여 눈 면봉과 문화를 처리하기 위해 한 손을 할당합니다. 케이지에서 마우스를 제거하고 왼쪽 눈이 노출된 측면에 위치하는 작업 표면 위에 놓습니다.장갑을 낀 손을 이소프로파놀로 스프레이하고 종이 타월로 말리십시오. 전용 미디어 핸들링 핸드로 표시된 BHI 마이크로 원심분리기 튜브를 언캡하고 두 개의 튜브를 랙에 다시 배치합니다. BHI에 면봉의 면 코팅 된 끝을 찍어, 다음 여분의 액체를 제거하고 제거하기 위해 내부 튜브에 대해 두 번 팁을 소용돌이동안 튜브에서 면봉을 철회.마우스 핸들링 핸드로 마우스를 목 덜미로 부드럽게 잡습니다. 반면에, 눈의 끝을 왼쪽 눈의 내측 결막 부위에 대고 놓습니다. 눈알을 가볍게 누르고, 낮은 눈꺼풀과 눈 사이의 세안 동작으로 면봉을 10번 움직이며 일정한 압력을 유지합니다.모피를 만지지 않고 면봉 끝을 삽입한 위치에 수직으로 부드럽게 제거합니다. 면봉 면면을 BHI 미디어로 라벨이 붙은 마이크로 원심분리기 튜브에 직접 놓습니다. 면봉된 눈에 눈방울을 바르는 다.원하는 경우 제어 시료를 위해 피부 또는 모피 면봉을 획득하여 각 면봉 사이에 적절하게 장갑을 살균합니다. 완료되면 마우스를 케이지로 반환합니다. 면봉이 얼음 위에 10-15분 동안 서게 한 다음 장갑을 낀 손을 살균하고 10회 회전을 위해 미디어에 팁을 혼합하면서 면봉을 제거합니다.튜브의 내벽에 대한 팁을 5회 회전하여 회전하여 면봉을 철회하고 생체 위험 용기에 폐기하십시오. 각 마우스에 대한 프로세스를 반복합니다. 37°C에서 1시간 동안 튜브를 정적으로 배양하여 샘플을 풍부하게 합니다.인큐베이션 하는 동안, 마우스 눈 면봉 또는 제어 면봉 당 하나의 실온 TSA 플레이트를 표시 하 고 반으로 분할. 인큐베이터에서 농축 된 샘플을 제거하고 얼음위에 놓습니다. 샘플을 짧게 소용돌이에 섞은 다음 샘플의 10 마이크로리터를 TSA 플레이트에 알리쿼트하고 플레이트를 기울여 스트립을 형성합니다.이 것을 두 번 반복합니다. 플레이트 분할 선의 반대편에는 각각 10개의 마이크로리터샘플이 있는 10개의 점을 만듭니다. 100접시가 건조되는 것을 방지하는 깨끗한 챔버에서 18시간, 2일, 4일 동안 37°C의 플레이트를 배양합니다.스트립의 식민지를 계산합니다. 형태학적으로 유사한 격리를 위해 면봉당 식민지 형성 단위를 계산합니다. 독특한 유기체가 스트립에 포착되지 않는 점을 살펴보십시오.시각화를 용이하게 하기 위해 중간 및 오른쪽 플레이트에서 박테리아를 조밀하게 도금했습니다. 전형적으로, 우리는 눈 면봉 판에 있는 훨씬 더 적은 박테리아를 관찰했습니다. 경우에 따라 눈 면봉이 회수 가능한 박테리아를 생성하지 못할 수 있습니다.C57 블랙 6 마우스에서 형태학적으로 다양한 분리가 있는 대표적인 눈 면봉판이 여기에 나와 있다. 각 고유의 격리에 대해, 식민지는 스트립에 계산되었고 상대적 풍요로움이 계산되고 플롯되었습니다. 미생물 특성화를 위해, 박테리아는 마스터 TSA 플레이트를 생성하기 위해 개별 마우스 눈 면봉 플레이트에서 수확되었다.성장이 나타났을 때, 추가 시험은 미생물을 특성화하거나 확인하기 위하여 실행되었습니다. 마스터 플레이트는 각각의 분리를 확장하기에 충분한 접종을 제공하는 데 사용되었습니다. 격리를 식별하기 위해 TSA 플레이트가 하룻밤 사이에 줄무늬와 배양되었고 MALDI-TOF MS가 수행되었습니다.여기에 표시된 분리물의 예는 연쇄상 구균 산산성소미니무스 및 에어로코커스 비리단으로 확인되었다. 기만 유기체의 상당히 다른 수준은 남성과 여성 C57 블랙 6 마우스에서 회복되었다. 연쇄상 구균 산성미니무스, 에어로코커스 비리단은 부정적인 황색 포도상 구균및 E.coli는 C57 블랙 6 N 마우스로부터 분리되었다.이 프로토콜을 시도할 때 면봉을 얇게 코팅하고 눈 면봉 단계에서 시간을 내어 최상의 결과를 얻을 수 있음을 명심하십시오. MALDI-TOF MS에 대한 접근이 제한되는 경우, 격리는 미생물 및 생화학적 테스트에 의해 확인될 수 있다.

conjunctival-commensal-isolation-and-identification-in-mice

Research

JoVE Journal

Immunology and Infection

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

에서 뇌 - 분리 된 백혈구의 분리 및 분석<em&gt; Plasmodium berghei</em&gt; ANKA에 감염된 마우스

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

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면역학 및 감염병학

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

MALDI-TOF 질량 분석기를 사용하여 혈액 배양액에서 그람 음성 박테리아의 빠른 확인

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

격리, 식별 및 쥐과 흉선 상피 세포의 정화

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

호중구의 분리 및 분석 치료제에 림프종 세포 감도에서 자신의 역할을 확인하는 방법

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Plasmodesmal 지역화 시퀀스 Planta에 단백질의 식별

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

살 모 넬 라 typhimurium의 절연-대 식 세포에서 Phagosomes를 포함 하

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

식별 및 Oligopotent 및 계보 커밋된 골수성 창시자 마우스 골 수에서 격리

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

절연 및 심장의 Extravascular 면역 세포의 식별

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

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

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

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

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

갤러리아 mellonella 구강 관리 모델 Study Commensal-Induced 타고 난 면역 반응에

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

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.