이 작업에 대한 재정 지원은 유럽 공동체 (ERC-STG INTRACELLTB 그랜트 n ° 260901, MM4TB 그랜트 n ° 260872), 기관 국립 드 레체쉬, 페더 (12001407 (D-AL) Equipex Imaginex BioMed), 및 지역 노르드 파스 칼레에 의해 제공되었다. 우리는 플랫폼 BICeL에서 가스파드 딜로이슨, 엘리자베스 베르크마이스터, 안토니노 본지오반니, 프랭크 라폰트의 기술적 도움을 감사하게 인정합니다.

1. 높은 처리량 게놈 와이드 siRNA 스크리닝
인간 유형-II 폐렴구체 모델 A549 세포주에서 수행된 스크리닝은 Mtb H37Rv를 통해 바이러스 성형 단백질(GFP)을 발현하는 감염 시 수행하였다. 이 절차는 그림 1A에설명되어 있습니다.
<ol><li>4 μM의 농도에 도달하기 위해 1 x siRNA 버퍼로 어머니 판 (96 웰 플레이트)에 저장된 말린 siRNA 라이브러리를 다시 중단한 다음 혼합물의 10 μl을 384 웰 딸 플레이트 (딸 플레이트 1)로 옮길 수 있습니다.</li>
	<li>딸 플레이트 1에 1x siRNA 버퍼 10 μl을 추가하여 siRNA를 2배 희석시 합니다. siRNA 재서스펜션 후, 플레이트는 벗겨질 수 있는 알루미늄 씰로 밀봉되어 -20°C이상 에서 최소 6개월 및 최대 2-3년까지 보관할 수 있지만, 저장 시간은 siRNA 라이브러리 제조업체의 권장 사항에 따라 달라질 수 있습니다.</li>
	<li>딸 접시 1에 siRNAs를 희석하여 딸 플레이트 2에 넣고 500 nM의 농도에 도달하십시오. siRNA 재서스펜션 후, 플레이트는 벗겨질 수 있는 알루미늄 씰로 밀봉되어 -20°C이상 에서 최소 6개월 및 최대 2-3년까지 보관할 수 있지만, 저장 시간은 siRNA 라이브러리 제조업체의 권장 사항에 따라 달라질 수 있습니다.</li>
	<li>사용하기 전에 실온에서 딸 플레이트 2를 해동하십시오.</li>
	<li>딸 플레이트 2에서 siRNA2.5 μl을 가져 와서 384 웰 분석 플레이트에 넣습니다.</li>
	<li>1.5단계에서 동일한 384-음분석 플레이트에서 각각의 우물에 2.5 μl 음수 및 양수 제어 siRNA를 추가합니다.</li>
	<li>1x D-PBS에서 트랜스페션 시약을 희석하여 각 우물에서 0.1 μl 형질 전환 시약을 제공하고 5분 동안 실온에서 희석된 형질 방출 용액을 미리 배양할 수 있는 충분한 용액을 산출합니다.</li>
	<li>형질 전환 시약/PBS 용액 혼합물의 7.5 μl을 분석 판에 각각 잘 넣고 실온에서 30분 동안 배양합니다.</li>
	<li>10% 태아 소 혈청(FBS)으로 보충된 RPMI 1640 배지에서 중단된 A549 세포(1,500세포/웰)의 40μl을 추가합니다. 5%CO2를함유하는 대기에서 37°C에서 3일 배양 기간 동안 세포를 유지한다. 이 세포는 매 24 시간 마다 분할, 따라서 대략 12,000 세포는 경질 후에 3 일 우물에 있습니다.</li>
	<li>2주 된 GFP 표현 Mtb H37Rv 문화를 D-PBS(MgCl2 및 CaCl2무료)로 씻어 내고 원심분리를 5분 동안 4,000 x g로 씻어 내고 세척을 폐기합니다. 이 단계를 3배 반복합니다(GFP-Mtb H37Rv 문화 조건의 경우 프로토콜 3참조).</li>
	<li>세균성 골을 10 ml의 RPMI 1640 배지에 10% FBS로 보충하고 세균성 골체가 퇴적물을 허용하도록 실온에서 1 시간 동안 데캔트를 보충합니다.</li>
	<li>세균성 상체를 수집하고 OD600(OD 600은 0.6-0.8 사이여야 함)과 GFP-형광(RFU 값) 및 마이크로플레이트 판독기를 사용하여 세균 농도를 결정한다. 동일한 조건에서 제조된 다른 배양에 대한 실험 전에 생성된 RFU 값 = f(CFU 값)를 표시하는 참조 회귀 선을 사용하여 서스펜션의 티터를 계산한다. 감염(MOI)의 복합성에 해당하는 2.4 x 106 박테리아/ml를 함유한 세균현탁액을 준비한다.</li>
	<li>384-음 분석 플레이트의 배지를 제거하고 갓 준비된 세균 현탁액 25 μl을 추가합니다.</li>
	<li>384-음분석 플레이트를 37°C에서 5%CO2를함유한 대기시 5시간 동안 배양한다.</li>
	<li>매체를 제거하고 10% FBS 3x로 보충된 RPMI 배지로 세포를 부드럽게 세척합니다.</li>
	<li>남은 세포외 세균을 죽이기 위해, 5%CO2를함유한 대기시 37°C에서 37°C에서 50 μg/ml를 함유한 신선한 RPMI-FBS 배지50 μl로 세포를 치료한다.</li>
	<li>중간 함유 아미카신을 제거하고 10% FBS로 보충된 신선한 RPMI 배지 50 μl을 추가합니다. 384-잘 분석 플레이트를 37°C에서 5일 동안 5%CO2를함유한 대기권에서 배양한다.</li>
	<li>이미지 수집 에 앞서 PBS (최종 농도 5 μg /ml)에 갓 준비 된 30 μg /ml의 10 μl을 추가하고 37 ° C에서 10 분 동안 배양하십시오.</li>
	<li>플레이트를 자동 공초점 현미경으로 로드합니다. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_1highres.jpg" src="/files/ftp_upload/51114/screenshot_1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_1highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a></li>
	<li>노출 매개 변수를 설정합니다. 방출 필터 450 nm및 GFP 형광을 이용한 발산 레이저 405 nm를 사용하여 DAPI 형광을 기록하여 방출 필터 520 nm를 가진 엑아시 레이저 488 nm를 사용한다. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-1highres.jpg" src="/files/ftp_upload/51114/screenshot_2-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-1highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-2highres.jpg" src="/files/ftp_upload/51114/screenshot_2-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-2highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a></li>
	<li>획득할 각 웰의 우물과 필드를 선택하여 레이아웃 및 하위 레이아웃 매개 변수라고 합니다. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-1highres.jpg" src="/files/ftp_upload/51114/screenshot_3-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-1highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-2highres.jpg" src="/files/ftp_upload/51114/screenshot_3-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-2highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a></li>
	<li>1.20 및 1.21 단계의 매개 변수를 사용하여 실험 파일을 생성하고 자동 수집을 실행합니다. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-1highres.jpg" src="/files/ftp_upload/51114/screenshot_4-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-1highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-2highres.jpg" src="/files/ftp_upload/51114/screenshot_4-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-2highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a></li>
	<li>이미지를 원격 서버로 전송합니다. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_5highres.jpg" src="/files/ftp_upload/51114/screenshot_5.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_5highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a></li>
	<li>이미지 분석 소프트웨어를 사용하여 이미지를 평가합니다. 픽셀 강도 특성알고리즘(도 4A)을사용하여 GFP 채널로부터 핵 검출 알고리즘 및 세균 영역을 사용하여 DAPI 채널에서 세포 핵을 검출한다.</li>
</ol>참고: 이 프로토콜은 세포 내 Mtb 성장에 대한 유전자 침묵의 효과를 연구하도록 최적화되어 있습니다. Mtb는 최적의 조건에서 매 20 시간마다 분할 느린 성장 박테리아입니다. 감염 후 5 일 후 세포 외 Mtb의 양은 세포 용해가 없는 상태에서 여전히 낮으며 분석의 품질에 영향을 미치지 않았습니다. 이 프로토콜은 광범위하게 방출되고 새로운 세포를 감염시킬 수 있는 Mycobacteria smegmatis 및 Escherichia coli 같이 급속한 성장 박테리아를 사용하여 siRNA 스크린에 적응하기 위하여 항생제 처리 및 잠복기 시간의 기간에, 최적화되어야 합니다.
2. 높은 처리량 화합물 스크리닝
Mtb H37Rv 감염 호스트 세포에 수행 된 스크리닝. 이 절차는 그림 1B에설명되어 있습니다.
<ol><li>DMSO 100%에서 용해된 화합물 라이브러리를 포함하는 384웰의 어머니 판을 해동한다. 10% FBS로 보충된 RPMI 1640 배지의 10 μl을 포함하는 384웰 딸 플레이트에서 화합물의 0.5 μl을 전송한다.</li>
	<li>2주 된 GFP 표현 Mtb H37Rv 문화를 D-PBS(MgCl2 및 CaCl2무료)로 씻어 내고 원심분리를 5분 동안 4,000 x g로 씻어 내고 세척을 폐기합니다. 이 단계를 3배 반복합니다(GFP-Mtb H37Rv 문화 조건의 경우 프로토콜 3참조).</li>
	<li>세균성 골을 10 ml의 RPMI 1640 배지에 10% FBS로 보충하고 세균성 골체가 퇴적물을 허용하도록 실온에서 1 시간 동안 데캔트를 보충합니다.</li>
	<li>세균상수체를 수집하고 마이크로플레이트 판독기를 사용하여 OD600(OD 600 은 0.6-0.8 사이여야 함)과 GFP 형광(RFU 값) 측정한다. 실험 전에 생성된 RFU 값 = f(CFU 값)를 표시하는 참조 회귀 선을 사용하여 서스펜션의 티터를 계산합니다. 일반적인 농도는 1 x 108 박테리아/ml입니다.</li>
	<li>수확 6 일 된 기본 인간 대식세포에서 4 x 105 세포/ml RPMI 1640 배지10% 태아 소 혈청 (FBS) 및 50 ng/ml 재조합 인간 M-CSF (인간 말초 혈액 모노사이클 세포 정화 및 대식세포 분화의 경우 프로토콜 4 참조).</li>
	<li>1-5에 이르는 다른 MOI에서 원액1차 세포를 37°C에서 2시간 동안 90rpm에서 가벼운 흔들림으로 현탁액하여 다른 MOI에서 바실리로 배양한다.</li>
	<li>감염된 세포를 350 x g에서 원심분리로 세척하여 세포외 박테리아를 제거합니다. 각 원심 분리 단계 후, 10% FBS로 보충된 RPMI 1640 배지에서 펠릿을 다시 놓습니다. 이 단계를 2배 반복합니다.</li>
	<li>50 μg/ml에서 10% FBS 및 아키카신으로 보충된 RPMI 1640 배지에서 감염된 세포를 중단하고 37°C에서 1시간 동안 가벼운 흔들림으로 현탁액을 배양한다.</li>
	<li>350 x g에서 원심분리에 의해 아키카신을 함유하는 세포 배양 배지를 제거하고 10% FBS 및 50 ng/ml 재조합 인간 M-CSF로 보충된 완전한 RPMI 1640 배지로 감염된 세포를 세척한다. 한 번 반복합니다.</li>
	<li>감염된 대식세포 현탁액의 40 μl을 2.1단계에서 동일한 분석 플레이트에 추가하여 이미 화합물 희석제의 10 μl을 함유하고 있습니다. 각 우물에서 DMSO의 최종 농도는 이제 1 %에 도달했습니다.</li>
	<li>5%CO2를함유한 대기에서 37°C에서 5일간 분석플레이트를 배양한다.</li>
	<li>세포가 구형극홍형염으로 살아있는 세포를 얼룩지게 합니다.</li>
	<li>플레이트를 자동 공초점 현미경으로 로드합니다. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_1highres.jpg" src="/files/ftp_upload/51114/screenshot_1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_1highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a></li>
	<li>노출 매개 변수를 설정합니다. 방출 필터 690 nm와 GFP 형광을 사용하여 방출 필터 640 nm와 GFP 형광을 사용하여 광적 형광을 기록 520 nm. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-1highres.jpg" src="/files/ftp_upload/51114/screenshot_2-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-1highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-2highres.jpg" src="/files/ftp_upload/51114/screenshot_2-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-2highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a></li>
	<li>획득할 각 웰의 우물과 필드를 선택하여 레이아웃 및 하위 레이아웃 매개 변수라고 합니다. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-1highres.jpg" src="/files/ftp_upload/51114/screenshot_3-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-1highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-2highres.jpg" src="/files/ftp_upload/51114/screenshot_3-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-2highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a></li>
	<li>2.14 및 2.15 단계의 매개 변수를 사용하여 실험 파일을 생성하고 자동 수집을 실행합니다. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-1highres.jpg" src="/files/ftp_upload/51114/screenshot_4-1.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-1highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a> <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-2highres.jpg" src="/files/ftp_upload/51114/screenshot_4-2.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-2highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a></li>
	<li>이미지를 원격 서버로 전송합니다. <img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_5highres.jpg" src="/files/ftp_upload/51114/screenshot_5.jpg"> <a href="https://www.jove.com/files/ftp_upload/51114/screenshot_5highres.jpg" target="_blank">큰 이미지를 보려면 여기를 클릭하십시오.</a></li>
	<li>이미지 분석 소프트웨어를 사용하여 이미지를 평가합니다. 픽셀 강도 속성알고리즘(그림 4B)을사용하여 GFP 채널에서 픽셀 강도 특성 알고리즘 및 세균 영역을 사용하여 극적 채널에서 셀 영역을 감지합니다.</li>
</ol>참고: 이 프로토콜은 형광 단백질(1개 우물/1 돌연변이)을 표현하는 돌연변이체에 의한 화합물을 대체하여 Mtb 돌연변이 라이브러리 스크리닝에 적용될 수있다(도 1C,브로딘 외 참조) 4).형광 돌연변이는 우물에서 먼저 시드된다 (잘 당 세균 현탁액의 20 μl). 박테리아는 그 때 세포 현탁액의 30 μl에 의해 복구됩니다. 원심분리후 350 x g에서 1분 동안, 플레이트는 5%CO2를함유하는 대기에서 37°C로 배양된다. 인큐베이션 시간 및 MOI는 분석에 의존합니다. 예를 들어, phagosome 산성화와 같은 초기 세포 이벤트의 시각화를 위해, 세포는 1-20에서 배열하는 MOI를 가진 2 시간 동안 감염될 수 있다. 리소솜은 리소트래커 염료를 2μM에서 1.5시간 동안 37°C에서 37°C로염색한 후 10% 포름알린 또는 4%의 파라포름데히드(PFA)로 고정된다. 공초점 이미지는 리소좀 검출 및 세포극 지역화4에적합한 알고리즘을 특징으로 하는 이미지 분석 스크립트를 사용하여 획득및 최종 적으로 분석된다.
3. 진균 결핵 H37Rv (GFP-H37Rv) 문화 조건을 표현하는 녹색 형광 단백질
장기 보관을 위해 GFP-H37Rv는 D-PBS(유리병당 약 1 x 108 균주)로 동결되었습니다.
<ol><li>미들브룩 OADC 농축 10%, 글리세롤 0.5%, 트웬-80 0.05%, 하이그로마이신 B(50 μg/ml)로 보충된 7H9 국물 중형 50ml를 함유한 Erlenmeyer 플라스크에서 GFP-H37Rv의 냉동 육수 바이알 1개를 재차 중단합니다.</li>
	<li>37 °C에서 8 일 동안 배양하십시오.</li>
	<li>GFP-H37Rv 배양의 OD600을 측정합니다.</li>
	<li>GFP-H37Rv 배양을 희석하여 미들브룩 OADC 농축10%, 글리세롤 0.5%, 트위엔-80 0.05%, 하이그로미신 B(50 μg/ml)로 보충된 신선한 7H9 국물 배지에서 OD 600 = 0.1을 얻습니다.</li>
	<li>GFP-H37Rv를 37°C에서 37°C에서 8일 더 배양한 후 분석에 사용한다.</li>
</ol>4. 전신 혈액 또는 버피 코트 준비에서 인간의 말초 혈액 단화 세포 정화
<ol><li>1%의 FBS를 함유한 1x D-PBS(MgCl 2 및 CaCl2에서 무료)로 혈액 파우치2x를희석시합니다.</li>
	<li>Ficoll 밀도 그라데이션 원심분리에 의해 단핵구를 400 x g에서 20분 동안 분리합니다.</li>
	<li>격리된 단핵구를 수집합니다.</li>
	<li>1x D-PBS(MgCl2 및 CaCl2에서무료)로 모노사이클 3x를 세척하여 실온에서 10분 동안 400 x g에서 원심분리로 1% FBS를 함유하고 있습니다.</li>
	<li>세포를 최대 1 x 107 셀/ml로 농축합니다.</li>
	<li>제조업체의 프로토콜에 따라 CD14 자성 구슬을 사용하여 단백구를 정화합니다(재료 참조).</li>
	<li>CD14-단핵구 정제 후, RPMI 1640에서 1.5 x 106 셀/ml의 세포를 종자 10% FBS와 40 ng/ml의 인간 대식세포 콜로니 자극 인자(hM-CSF)를 보완하고 5%CO2를함유한 대기 중 37°C에서 4일 동안 배양하였다.</li>
	<li>4일 후 신선한 RPMI 1640으로 배지를 10% FBS와 40 ng/mlhM-CSF로 보완하고 5%CO2를함유한 대기 중 37°C에서 2일간 배양하였다.</li>
	<li>6 일 후, 세포는 분석에 사용할 수 있습니다.</li>
</ol>

<ol>
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K., Cechetto, J., Brodin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-content+imaging+of+Mycobacterium+tuberculosis-infected+macrophages:+an+in+vitro+model+for+tuberculosis+drug+discovery.">High-content imaging of Mycobacterium tuberculosis-infected macrophages: an in vitro model for tuberculosis drug discovery.</a> Future Med. Chem. 2, 1283-1293 (2010).</li><li>Brodin, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+content+phenotypic+cell-based+visual+screen+identifies+Mycobacterium+tuberculosis+acyltrehalose-containing+glycolipids+involved+in+phagosome+remodeling.">High content phenotypic cell-based visual screen identifies Mycobacterium tuberculosis acyltrehalose-containing glycolipids involved in phagosome remodeling.</a> PLoS Pathog. 6, (2010).</li><li>Bermudez, L. 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이해 상충이 선언되지 않았습니다.

우리는 여기에 GFP 표현 Mtb H37Rv 균주를 사용하여 표현 형광 숙주 세포를 감염시키는 표현적 분석에 필요한 방법을 설명, 이는 높은 함량 / 높은 처리량 스크린에 적합하게. 이 프로토콜은 광범위한 화합물, 형광 프로브 및 Mtb 돌연변이체에 적용될 수 있습니다. 위에서 설명한 각 프로토콜에 대해 이미지 수집 전에 고정 및 면역 라벨링 단계를 수행할 수 있습니다. 우리는 이미지를 얻기...

지난 몇 년 동안 보고된 신흥 및 재발하는 전염하는 병원균 중, 진균 결핵(Mtb)은 2011년에 140만 명의 사망자와 870만 건의 새로운 감염을 담당하는 두드러진 장소를 보유하고 있습니다(글로벌 결핵 보고서 2012, www.who.int/topics/tuberculosis/en/). 다약물 치료의 가용성에도 불구하고, 감염된 사람들의 수는 여전히 증가하고 다중 약물 내성 (MDR) 뿐만 아니라 광범위하게 약물 내성 (XDR) Mtb는 빠르게 전 세계적으로확산1. 더욱이, Mtb 항원의 존재를 고려할 때, 전 세계 인구의 3분의 1이 Mtb에 의해 잠잠하게 감염된 것으로 간주된다는 것이 분명합니다. 따라서 Mtb과 싸우는 새로운 수단이 절실히 필요합니다. 이러한 맥락에서, 우리는 자동화된 공초점 형광 현미경3에의하여 숙주 세포로 Mtb 침입 및 곱셈을 감시에 의존하는 체외 시각적 표현체 분석법을 개발하였다. 384웰 마이크로티터 플레이트에서 자동 이미지 수집 및 분석과 결합하여 분석체의 적응은 화합물, siRNAs 및 세균 돌연변이의 중간 규모의 라이브러리의 고함량/고처리량 스크리닝(HC/HTS)을 허용했습니다. 이 표현성 분석에 게놈 넓은 RNAi 라이브러리의 검사는 따라서 Mtb 인신 매매 및 세포 내 복제에 관련된 주요 호스트 요인뿐만 아니라 결핵 바실러스에 의해 악용 호스트 경로의 용해를 가능하게했다. 이 특정 현상 분석의 또 다른 적응은 Mtb 내 phagosomal 지속성에 필수적인 세균인의 식별을 위한 것이었습니다. 예를 들어, phagosome 성숙의 체포는 대식세포에서 Mtb의 생존 그리고 복제를 용이하게 하는 주요 기계장치 의 한으로 간주됩니다. 형광 표지산산구획에서 Mtb 녹아웃 돌연변이체의 세포외 국소화 모니터링은 생존 과정에 관여하는 세균 유전자의 식별을허용4. 마지막으로, Mtb의 고함량 이미징은 세포내 세균 성장3과같은 다양한 현상을 억제하기 위한 약물 효율을 정량화하는 우수한 방법을 제공한다. 전부, 높은 처리량 표현분석의 이 모형은 결핵에 대하여 약 발견을 가속화하고 이러한 다른 접근에 의해 집합된 데이터는 Mtb에의해 발휘된 호스트 조작의 더 나은 이해에 기여합니다.

<table border="0" cellpadding="0" cellspacing="0" width="1010">
	<colgroup>
		<col />
		<col /> 
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">&#181;clear-plate black, 384 well</td>
			<td style="width:160px;">Greiner bio-one</td>
			<td style="width:175px;">781091</td>
			<td style="width:388px;">127.8/86/15 MM with Lid, TC treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">CellCarrier 384 well plate</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;">6007550</td>
			<td>Black, Clear Bottom, with Lid, TC treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">V-bottom white , 384 well-plate</td>
			<td style="width:160px;">Greiner bio-one</td>
			<td style="width:175px;">781280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">sealing tape, breathable, sterile</td>
			<td style="width:160px;">Corning</td>
			<td style="width:175px;">3345</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Lipofectamine RNAiMax</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">13778150</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Dimethyl sulfoxide</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">34943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">RPMI 1640 + GlutaMAX-I</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">61870-010</td>
			<td>Cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">D-PBS 1X [-]MgCl2/[-]CaCl2</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">14190-094</td>
			<td>Dulbecco&#39;s Phosphate Salin Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">D-PBS 1X [+]MgCl2/[+]CaCl2</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">14190-091</td>
			<td>Dulbecco&#39;s Phosphate Salin Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Fetal bovine serum</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">2610040-79</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">FICOLL PAQUE PLUS</td>
			<td style="width:160px;">DUTSCHER</td>
			<td style="width:175px;">17-1440-03</td>
			<td>Ficoll for Peripherical Blood Monocyte Cells purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">CD14 MicroBeads, human</td>
			<td style="width:160px;">Miltenyi</td>
			<td style="width:175px;">130-050-201</td>
			<td>Purification of CD14+ Monocytes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Human M-CSF, premium gr. (1000 &#956;g)</td>
			<td style="width:160px;">Miltenyi</td>
			<td style="width:175px;">130-096-493</td>
			<td>Macrophage Colony Stimulating Factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">LS Columns</td>
			<td style="width:160px;">Miltenyi</td>
			<td style="width:175px;">130-042-401</td>
			<td>Columns for CD14+ Monocytes isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Tween 80</td>
			<td style="width:160px;">Euromedex</td>
			<td style="width:175px;">2002-A</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Glycerol high purity </td>
			<td style="width:160px;">Euromedex</td>
			<td style="width:175px;">50405-EX</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;"> Middlebrook OADC enrichment</td>
			<td style="width:160px;">Becton-Dickinson</td>
			<td style="width:175px;">211886</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">7H9</td>
			<td style="width:160px;">Becton-Dickinson</td>
			<td style="width:175px;">W1701P</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Versene 1X</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">15040033</td>
			<td>Non enzymatic cell dissociation solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">DAPI</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">D1306</td>
			<td>Nuclei dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hoechst 33342</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">H3570</td>
			<td>Nuclei dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Syto60</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">S11342</td>
			<td>Nuclei/cytoplasm dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Formalin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">HT5014</td>
			<td>Cell fixation solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">siRNA targeting Dectin-1</td>
			<td style="width:160px;">Santa-Cruz</td>
			<td style="width:175px;">sc-63276</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">*siGenome* Non targeted siRNA pool </td>
			<td style="width:160px;">Dharmacon</td>
			<td style="width:175px;">D-001206-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Rifampicin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">R3501</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Isoniazid (INH)</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">I3377-50G</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hygromycin B</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">10687-010</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Amikacin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">A1774</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Automated Confocal Microscope OPERA</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;"> </td>
			<td>Image acquisition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Columbus 2.3.1 Server Database</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;"> </td>
			<td>Data transfert, storage and analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Acapella 2.6 software</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;"> </td>
			<td>Image-based analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">GraphPad Prism5 software</td>
			<td style="width:160px;">GraphPad </td>
			<td style="width:175px;"> </td>
			<td>Statistical analysis</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:287px;">Excel 2010</td>
			<td style="width:160px;">Microsoft</td>
			<td style="width:175px;"> </td>
			<td>Statistical analysis</td>
		</tr>
	</tbody>
</table>

a microscopic phenotypic assay for the quantification of intracellular mycobacteria adapted for high-throughput/high-content screening

치료와 백신의 가용성에도 불구하고 결핵 (TB)은 세계에서 가장 치명적이고 광범위한 세균 감염 중 하나입니다. 수십 년 이후, 다중 및 광범위 하 게 약물 저항 하는 긴장의 갑작스런 버스트는 결핵의 제어에 대 한 심각한 위협. 따라서 결핵, 진균 결핵(Mtb)의 원인제에 중요한 새로운 표적과 경로를 식별하고 결핵 치료제가 될 수있는 새로운 화학물질을 찾는 것이 필수적이다. 한 가지 접근법은 건초 더미에서 바늘을 검색 할 수 있도록 대규모 라이브러리의 유전 및 화학 적 스크린에 적합한 방법을 설정하는 것입니다. 이를 위해, 우리는 자동 공초점 현미경 검사를 사용하여 형광 표지 숙주 세포 내에서 형광 표지 Mtb의 검출에 의존하는 phenotypic 분석법을 개발했습니다. 이러한 시험관 내 분석법은 Mtb의 식민지화 공정을 숙주로 정량화할 수 있으며, siRNA-, 화학 화합물 또는 Mtb 돌연변이 라이브러리의 스크린에 적합한 384웰 마이크로플레이트 포맷에 최적화되었다. 그런 다음 이미지는 다중 파라메트릭 분석을 위해 처리되며, 이는 숙주 세포 내의 Mtb의 발병기에서 추론을 제공합니다.

높은 처리량 게놈 와이드 siRNA 스크리닝
Mtb는 체외뿐만 아니라 여러 가지 다른 폐 상피 세포에서 면역 세포를 식민지화 할 수 있습니다. 예를 들어, Mtb는인간 형 II 폐렴구균5-7의모델로 일반적으로 사용되는 A549 상피 세포를 감염시키고 손상시킬 수 있다. Dectin-1은 A549 세포에서 세포 내 진균 성장에 Mtb uptake, 프로염증 반응 및 항균 효과에 관여?...

Watch this Scientific Journal Video about A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening at JoVE.com

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

여기서는 작은 간섭 합성 RNA(siRNA), 화학 화합물 및 진균 결핵 돌연변이 라이브러리의 고처리량/고함량 스크린에 적용되는 표현성 분석을 설명합니다. 이 방법은 자동 공초점 현미경 검사를 사용하여 형광으로 표지된 숙주 세포 내의 형광표지 진균 결핵의 검출에 의존합니다.

고처리량/고함량 스크리닝에 적용된 세포내 진균의 정량화를 위한 현미경 표티픽 분석

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

a-microscopic-phenotypic-assay-for-quantification-intracellular

Research

JoVE Journal

Immunology and Infection

7.6K 조회수.  Université de Lille. 여기서는 작은 간섭 합성 RNA(siRNA), 화학 화합물 및 진균 결핵 돌연변이 라이브러리의 고처리량/고함량 스크린에 적용되는 표현성 분석을 설명합니다. 이 방법은 자동 공초점 현미경 검사를 사용하여 형광으로 표지된 숙주 세포 내의 형광표지 진균 결핵의 검출에 의존합니다.

비디오: A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

Mycobacteria의 가능성을 측정하는 기능 전체 혈액 분석은 리포터 - 진 BCG 또는 M. TB (BCG 태그 사용 럭스 / M. TB 럭스)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

연구

면역학 및 감염병학

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

세포에 붙어 Antileishmanial 검사에 대한 기생충 구조 및 변화 분석<em&gt; Leishmania donovani</emTHP1 인간 급성 백혈병 Monocytic 셀 라인에서&gt; Amastigotes

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not associated with the protein in nature. Literature has provided numerous examples regarding the implementation of directed evolution to successfully alter molecular specificity and catalysis1. The primary advantage of utilizing directed evolution instead of more rational-based approaches for molecular engineering relates to the volume and diversity of variants that can be screened2. One possible application of directed evolution involves improving structural stability of bacteriolytic enzymes, such as endolysins. Bacteriophage encode and express endolysins to hydrolyze a critical covalent bond in the peptidoglycan (i.e. cell wall) of bacteria, resulting in host cell lysis and liberation of progeny virions. Notably, these enzymes possess the ability to extrinsically induce lysis to susceptible bacteria in the absence of phage and furthermore have been validated both in vitro and in vivo for their therapeutic potential3-5. The subject of our directed evolution study involves the PlyC endolysin, which is composed of PlyCA and PlyCB subunits6. When purified and added extrinsically, the PlyC holoenzyme lyses group A streptococci (GAS) as well as other streptococcal groups in a matter of seconds and furthermore has been validated in vivo against GAS7. Significantly, monitoring residual enzyme kinetics after elevated temperature incubation provides distinct evidence that PlyC loses lytic activity abruptly at 45 &deg;C, suggesting a short therapeutic shelf life, which may limit additional development of this enzyme. Further studies reveal the lack of thermal stability is only observed for the PlyCA subunit, whereas the PlyCB subunit is stable up to ~90 &deg;C (unpublished observation). In addition to PlyC, there are several examples in literature that describe the thermolabile nature of endolysins. For example, the Staphylococcus aureus endolysin LysK and Streptococcus pneumoniae endolysins Cpl-1 and Pal lose activity spontaneously at 42 &deg;C, 43.5 &deg;C and 50.2 &deg;C, respectively8-10. According to the Arrhenius equation, which relates the rate of a chemical reaction to the temperature present in the particular system, an increase in thermostability will correlate with an increase in shelf life expectancy11. Toward this end, directed evolution has been shown to be a useful tool for altering the thermal activity of various molecules in nature, but never has this particular technology been exploited successfully for the study of bacteriolytic enzymes. Likewise, successful accounts of progressing the structural stability of this particular class of antimicrobials altogether are nonexistent. In this video, we employ a novel methodology that uses an error-prone DNA polymerase followed by an optimized screening process using a 96 well microtiter plate format to identify mutations to the PlyCA subunit of the PlyC streptococcal endolysin that correlate to an increase in enzyme kinetic stability (Figure 1). Results after just one round of random mutagenesis suggest the methodology is generating PlyC variants that retain more than twice the residual activity when compared to wild-type (WT) PlyC after elevated temperature treatment.

A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.

내열 Bacteriolytic 효소의 감독 진화에 대한 새로운 검사 방법

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

세대와의 멀티 표현형 높은 콘텐츠 상영<em&gt; Coxiella burnetii의</em&gt; 트랜스포존 돌연변이

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

마이코 박테리아의 세미 양적, 중간 처리량 열거의 숯 한천 레사 주린 분석의 시각화

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

개 호중구 세포 외 트랩 릴리스의 정량화를위한 ​​간단한 형광 분석

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

작은 분자의 높은 처리량 스크리닝 적합한 형광 계 분석 림프구

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

세포 내 타겟팅 억제제의 효능 및 세포 독성 검사를위한 시스템<em&gt; 결핵균</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

대 식 세포 안의 세포내 성장의 정량화는 빠르고 Leishmania 기생충의 독성을 평가 하기 위한 신뢰할 수 있는 방법

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

높은 처리량, 높은 콘텐츠, 액체 기반의 C. 선 충 Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...