Name: high throughput, real-time, dual-readout testing of intracellular antimicrobial activity and eukaryotic cell cytotoxicity
Uploaded: 2016-11-16T15:00:00
Description: Watch this Scientific Journal Video about High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity at JoVE.com

이 논문에보고 된 연구 내용은 전적으로 저자의 책임이며 JEK하는 보너스 번호 R01AI099122에서 알레르기 및 국립 보건원 (National Institutes of Health)의 감염증의 국립 연구소에 의해 지원되었다 반드시 국립 연구소의의 공식 견해를 대변하지 않습니다 건강. 우리는 ICCB - 롱 우드의 검사 시설 및 / 또는 국가 검사 실험실을위한에서 제니퍼 스미스, 데이비드 Wrobel, 스와 치앙마이, 더그 홍수, 숀 존스턴, 제니퍼 Nale, 스튜어트 Rudnicki, 폴 연의, 리처드 시우, 레이첼 소장에게 감사의 말씀을 우수의 뉴 잉글랜드 지역 센터 디펜스의 개발과 높은 처리량 심사 분석의 성능을 자신의 도움 (U54AI057159에서 지원) 감염증을 신흥. 우리는 또한 원고에 도움이 의견 케네스 P. 스미스에게 감사의 말씀을 전합니다.

1. 실시간 세포 내 성장과 진핵 세포 세포 독성 분석 <ol><li> 제조 된 숙주 세포 (J774A.1 세포) <ol><li> 9 %의 철 보충 혈청과 RPMI 1640에서 서스펜션 문화 J774A.1 뮤스의 musculus 대 식세포 유사 세포. 조직 배양 플라스크에 처음 통로. 세포 배지 15 ㎖에 75 ㎠로 조직 배양 플라스크에서 컨 플루 언트하게 한 후 15 ㎖의 조직 배양 플라스크로 복귀 50 ml를 전달하는 매체의 동일한 유형, 65 ml의 긁?...

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우리는 세포 내 박테리아의 성장 및 숙주 세포 독성의 동시 검출을위한 실시간 분석을 설명한다. 프로토콜에서 몇 가지 중요한 단계가 있습니다. 첫째, 강력한 분석 성능, 세균 및 세포 독성 판독 사이에 충분한 스펙트럼 분리가 있어야합니다. 이러한 분리는 루시 페라 제 오페론 기자와 형광 DNA 결합 염료의 조합에 대한 고유이다. 그러나, 우리의 경험 (표 1-3,도 2),<...

세포 내 구획에 일시적으로 증가 또는 상주 병원균은 치료 근절하기 어렵다. 의무 또는 상대적으로 이러한 레지오넬라 뉴모 필라, Coxiella burnetii의, 브루셀라 종으로 세포 내 병원균을 의무. , Francisella의 야토 및 진균 종. 종종 달에 몇 년의 범위 일 수있다 치료를위한 항생제 치료의 과정을 연장 필요합니다. 또한, 세포 외 병원균은 일시적으로 세포 내 틈새 시장을 점유하고이 방법으로 항생제 치료의 일반적인 과정에 의해 허가를 탈출하고 나중에 악성 감염의 새로운 라운드를 시작합니다 나타날 수 있습니다. 황색 포도상 구균 (1) 및 uropathogenic 장내 세균 2,3 감염이 점점 인식 예입니다. 따라서, 근본적으로 신약 개발 목표는 세포 내 구획 침투 신규 항생제를 확인하는 것이다. 최적의 치료는 빨리 근절하기세포 생물 및 하위 억제 항균 노출을 통해 저항의 개발을 방지 특히 바람직하다. 이를 위해 모델 병원체, 레지오넬라 뉴모의 세포 성장을 표적 세포 - 침투제 항균제를 식별하기 위해 고 처리량 스크리닝 기술을 개발했다. 4 이전 임상 관찰은 표준 항생제 감수성 검사가 정확하게이 생물에 대한 생체 내 치료 효과에 예측하지 않았 음을 나타냅니다. 이러한 β - 락탐 및 아미노 글리코 사이드와 같은 항균제의 주요 클래스, axenically 성장 레지오넬라 균에 대해 매우 효과적이지만, 충분히 레지오넬라 균이있는 세포 내 구획에 침투하지 않기 때문에 5는 특히이이었다. 5,6 나중에 증거가 기술적으로 더 복잡한 세포 성장 분석을 효과적으로 임상 적 효능을 예측하는 것이 좋습니다. (7) </sup&gt; 불행하게도, 이러한 분석은 집락 형성 단위 열거 위해 상이한 시간 점에서 용해 될 항균제로 처리 감염된 대 식세포를 요하는 매우 힘든 종점 분석법이었다. 이러한 분석은 대규모로 수행하는 것은 비실용적이고 높은 처리량 약물 발견에 부적합하다. 따라서, 우리는 세포 내 세균 성장의 실시간 측정을위한 기술을 개발했다. 6이 박테리아 염색체에 4 형광 단백질을 (이전에 기술, 1 세대 분석) 중 세균 루시퍼 라제 오페론 (8)의 통합을 통해 변형 된 박테리아 균주의 사용을 통해 구 기자 (여기 한 제 2 세대, 직교 분석 등)를 달성했다. 이러한 방식으로, 발광 또는 형광 신호 세균 수의 대용 실시간 판독을 제공한다. 그러나, 이러한 특성은 세포 내 infectio의 주요 교란 요인을 해결하지n 개의 분석, 숙주 세포에 오프 대상 효과. 구체적으로, 숙주 세포의 죽음은 본질적으로 세포의 성장을 제한하고, 항균 효과의 위양성 식별 리드. 심사 라이브러리의 많은 화합물은 진핵 세포 독성, 이러한 오탐 (false positive)이 후속, 해결을위한 엔드 포인트 세포 독성 분석의 큰 숫자를 필요로 사실 항균제를 압도 할 것입니다. 따라서, 동시에 진핵 세포 생존율 및 세포 성장을 평가 할 수 있도록 큰 관심이었다. 특히, 비 생존 진핵 세포의 특성은 세포막 무결성의 손실이다. 세포막의 투과성을 테스트 프로브 따라서 세포 생존 능력을 평가하기 위해 사용될 수있다. 우리는 이전에 액세스 사균의 핵 DNA를 염색하기 위해 추정되는 세포막 - impermeant, 형광 DNA 결합 염료의 일련의 기능을 특징으로한다. 4 핵 DNA 결합에,이 염료는 quantu에서 큰 증가를 표시배경 솔루션 형광 이상 증가 신호의 결과로 m 형광 수율. 따라서, 이러한 염료는 진핵 세포 사멸의 정량적 판독을 제공 하였다. (4) 특히, 우리는 여러 가지가 J774의 대 식세포와 연장 공동 배양하는 동안 자신 무독성 것으로 나타났다. 초기 감염 기간 동안 첨가하면, 그것들은 실시간 현미경 마이크로 플레이트 형광 계에 의해 측정 또는 관찰 할 수있는 진핵 세포 사멸의 형광 판독 값을 제공 하였다. 따라서, 세균 리포터 무독성, 막 impermeant 사용을 결합하여, DNA 결합 염료, 우리는 세균 부하 동시에 진핵 세포의 세포 독성을 동시에 측정 할 수있는 단순하고 비파괴 실시간 분석을 개발할 수 있었다. 이 분석은 우리의 세포 성장을 억제 할 수있는 능력을 위해 ~을 포함하여 기능적지지 않은 활동 (250) 항균제 및&gt; 240,000 작은 분자를 384 웰 플레이트 형식으로 화면 ~ 10,000 알려진 생체 활성제 할 수있다레지오넬라 뉴모 동시에 각 화합물 진핵 세포 독성 데이터를 생성하는 동안. 레지오넬라 균의 세포 성장에 대한 알려진 항생제 (6) 우리의 분석은 지금까지이 유형의 가장 포괄적 인 탐사했다. 6 병용 할 때 우리의 분석 포맷의 효율에 기초하여, 우리는 다음에 공지 된 항균제의 잠재적 상승 효과를 탐구. 가장 일반적인 시너지 테스트 중 하나, 소위 바둑판 분석은 표준 적 둘 이상의 항균제의 2 배 희석액의 조합 효과를 평가함으로써 수행된다. 두개 이상의 항 미생물제를 별도로 적용 각각의 효과의 합보다 함께 적용될 때 이러한 분석에서 10 시너지는 큰 효과의 관찰에 의해 정의된다. 참고로, 지금까지 만 집중과 선택적 시너지 시험은 세포 내 레지오넬라 뉴모 필라에 대해 수행되었다 </em&gt; 필요한 조합 순열을 곱한 기존의 엔드 포인트 분석에 포함 된 큰 노력 때문이다. 시너지 테스트를 용이하게하기 위해 디지털 자동 분배 기술 (6)과 조합하여 실시간 세포 성장 / 진핵 세포 독성 분석에 사용했다. 이 자동화는 DMSO 또는 수용액 단독 또는 384 웰 형식의 조합에 용해 화합물의 연속 희석을 분배하는 우리를 허용. (11) 또한, 이러한 강력한 액체 핸들링 기술은 용이하게 더 높은 해상도를 수행하라고 허가 제곱근 -의 두 (대신 표준보다 낮은 해상도, 두배) 우리 이차원 특이성 높은 수준을 달성하기 위해 희석 조합을 바둑판 시너지 분석. 이 배 희석 시리즈 12을 사용하는 경우이 해결 재현성에 대한 시너지 분야에서 문제를 해결에 특히 유용했다. 마지막으로, 우리의 분석은 정량적도 THER했다는 efore 억제의 그라데이션을 측정 하였다. 결과적으로, 분석은 성장 억제 비슷한 수준으로 조합 농도 isocontours 연결되는 isocontour isobolograms의 발현 억제 내용 전체를 캡처. 6이 묘화 전략은 조합 적 투여 량 - 반응 곡선을 가시화시켰다. 우리의 방법론을 설명하기 위해, 우리는 이러한 분석을 수행하기위한 우리의 프로토콜을 설명하고 대표적인 결과를 보여줍니다.

<table border="0" cellpadding="0" cellspacing="0" width="1458">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">J774A.1 cells</td>
			<td style="width:248px;">American Type Culture Collection</td>
			<td style="width:161px;">TIB-67</td>
			<td style="width:627px;">Host cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">ACES</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">A9758&#160;</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Yeast extract, ultrafiltered</td>
			<td style="width:248px;">Becton-Dickinson/Difco</td>
			<td style="width:161px;">210929</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium; lower grades may cause impaired growth and/or alter sensitivity of Legionella to growth inhibitors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Alpha-ketoglutaric acid, monopotassium salt</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">K2000</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sodium pyruvate</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">P5280</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Potassium phosphate, dibasic</td>
			<td style="width:248px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">P288-500</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">L-cysteine</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">C-7755</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Ammonium iron(III) citrate</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">F5879</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium; ferric pyrophosphate may be used instead but is more difficult to weigh accurately</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Potassium hydroxide solution, concentrated</td>
			<td style="width:248px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">SP236-500</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Deonized water</td>
			<td style="width:248px;">N/A</td>
			<td style="width:161px;">N/A</td>
			<td style="width:627px;">For making buffered charcoal yeast extract agar and buffered yeast extract medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:423px;">Thymidine (tissue culture grade)</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">T1895</td>
			<td style="width:627px;">For supplementing both RPMI 1640 and buffered yeast extract agar/medium &#8212; lower grade thymidine may be used for the latter, but may cause impaired cell growth and/or cell death in RPMI 1640</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">RPMI 1640, standard formulation</td>
			<td style="width:248px;">Corning via Thermo Fisher Scientific</td>
			<td style="width:161px;">10-040-CV</td>
			<td style="width:627px;">For growing J774A.1 cells prior to plating; includes 2 mM L-glutamine</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">RPMI 1640 lacking phenol red</td>
			<td style="width:248px;">Corning via Thermo Fisher Scientific</td>
			<td style="width:161px;">17-105-CV</td>
			<td style="width:627px;">For plating J774A.1 cells in 384 well dishes (not suitable for growth prior to plating); also lacks L-glutamine &#8212; supplement to 2 mM before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">L-glutamine, 200 mM in 0.85% NaCl (tissue culture grade)</td>
			<td style="width:248px;">HyClone via Thermo Fisher Scientific</td>
			<td style="width:161px;">SH30034.02</td>
			<td style="width:627px;">For supplementing RPMI 1640 lacking L-glutamine, to 2 mM final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Iron-supplemented calf serum</td>
			<td style="width:248px;">Gemini Bioproducts</td>
			<td style="width:161px;">100-510</td>
			<td style="width:627px;">For supplementing RPMI 1640, to 9.1% final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Trypan Blue solution</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">T8154</td>
			<td style="width:627px;">For staining for J774A.1 cell death determination while counting cell density</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:423px;">SYTOX Green, 5 mM solution in DMSO</td>
			<td style="width:248px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">S7020</td>
			<td style="width:627px;">For staining for J774A.1 cell death determination by fluorescence reading or epifluorescence microscopy (in conjunction with orange-red or far red fluorescent bacteria). Use at 125 nM final concentration.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">GelRed, 10000X solution in water</td>
			<td style="width:248px;">Biotium</td>
			<td style="width:161px;">41003</td>
			<td style="width:627px;">For staining for J774A.1 cell death determination by epifluorescence microscopy (in conjunction with green fluorescent bacteria). Use Gel Red&#160; at 1X final concentration.&#160;&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Cell culture incubator</td>
			<td style="width:248px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">13-255-26</td>
			<td style="width:627px;">For incubation of J774A.1 cells (both before and after infection); can also be used for incubation of bacteria, or a standard atmosphere incubator can be used instead)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Orbital shaker</td>
			<td style="width:248px;">BellCo Glass</td>
			<td style="width:161px;">7744-01010</td>
			<td style="width:627px;">For shaking incubation of J774A.1 cells before infection; fits inside cell culture incubator; includes shaker base 7744-01000 and tray 7740-01010 (these are also available separately)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Shaker flasks (250 ml)</td>
			<td style="width:248px;">ChemGlass Life Sciences</td>
			<td style="width:161px;">CLS-2038-04</td>
			<td style="width:627px;">For shaking incubation of J774A.1 cells before infection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Shaker clamps for flasks (250 ml)</td>
			<td style="width:248px;">BellCo Glass</td>
			<td style="width:161px;">7744-16250</td>
			<td style="width:627px;">For shaking incubation of J774A.1 cells before infection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Shaker flasks (1000 ml)</td>
			<td style="width:248px;">ChemGlass Life Sciences</td>
			<td style="width:161px;">CLS-2038-07</td>
			<td style="width:627px;">For shaking incubation of J774A.1 cells before infection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Shaker clamps for flasks (1000 ml)</td>
			<td style="width:248px;">BellCo Glass</td>
			<td style="width:161px;">7744-16100</td>
			<td style="width:627px;">For shaking incubation of J774A.1 cells before infection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Sponge foam caps for flasks (250 ml - 1000 ml)</td>
			<td style="width:248px;">ChemGlass Life Sciences</td>
			<td style="width:161px;">CLS-1490-038</td>
			<td style="width:627px;">For shaking incubation of J774A.1 cells before infection; reduces risk of contamination relative to standard metal caps</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">MultiDrop Combi programmable multichannel peristaltic pump</td>
			<td style="width:248px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">5840300</td>
			<td style="width:627px;">For dispensing J774A.1 cells, medium, and bacterial suspension containing fluorophores to large numbers of 384 well dishes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Combi standard bore manifold</td>
			<td style="width:248px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">24072670</td>
			<td style="width:627px;">Default predispense volume of 20 ml is insufficient to compensate for settling &#8212; increase to 80 ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">White 384 well dishes treated for tissue culture</td>
			<td style="width:248px;">Corning</td>
			<td style="width:161px;">3570</td>
			<td style="width:627px;">For reading luminescence and fluorescence; Greiner catalog # 781080 also tested successfully</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">DMSO (tissue culture grade, in sealed ampoules)</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">D2650</td>
			<td style="width:627px;">For dissolving positive control and test compounds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Azithromycin</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">PHR1088</td>
			<td style="width:627px;">Antibiotic positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Saponin (from Quillaja bark)</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:161px;">S-4521</td>
			<td style="width:627px;">Cytoxicity positive control</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Multichannel pipettor</td>
			<td style="width:248px;">Thermo Fisher Scientific</td>
			<td style="width:161px;">Finnpipette</td>
			<td style="width:627px;">For transfer of fixed amounts of positive control compounds; pipettor must have digital dispensing with detents to enable repetitive fixed volume dispensing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Epson pin transfer robot</td>
			<td style="width:248px;">Epson/ICCB-L</td>
			<td style="width:161px;">(Custom equipment)</td>
			<td style="width:627px;">For transfer of fixed amounts of test compounds from library arrays</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">&#160;D300 digital dispensing system</td>
			<td style="width:248px;">Hewlett-Packard via Tecan</td>
			<td style="width:161px;">D300</td>
			<td style="width:627px;">For transfer of variable amounts of test compounds ranging from 11 picoliters to 10 microliters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">T8+ cartridges for D300 digital dispensing system</td>
			<td style="width:248px;">Hewlett-Packard via Tecan</td>
			<td style="width:161px;">T8+</td>
			<td style="width:627px;">For dispensing test compounds</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:423px;">EnVision multi-mode plate reader</td>
			<td style="width:248px;">Perkin-Elmer</td>
			<td style="width:161px;">(Contact manufacturer)</td>
			<td style="width:627px;">For optimal detection of SYTOX Green fluorescence, use excitation filter 485/14, emission filter 535/25, and dichroic mirror 505 nm, with selection of minimum gain and transmittance, and &#8220;high concentration mode. For luminescence detection, use the &#34;USLUM&#34; protocol for high-sensitivity detection. For mNeptune2 detection, use excitation filter 600/8, emission filter 665/7.5, and dichroic mirror 658 nm, with selection of gain and transmittance to achieve the highest maximum signal possible without saturating the photomultiplier.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:423px;">Epifluorescence microscope with computer-connected digital camera</td>
			<td style="width:248px;">Nikon</td>
			<td style="width:161px;">Ti</td>
			<td style="width:627px;">For live cell imaging; any standard fluorescent microscope can substitute, with phase contrast or DIC optics, capable of imaging green (fluorescein), orange-red to red (Texas Red), and far-red (Cy5) fluorescence, with 100X oil objective for highest resolution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:423px;">Glass-bottom tissue culture dishes</td>
			<td style="width:248px;">MatTek Corporation</td>
			<td style="width:161px;">P35G-1.5-20-C</td>
			<td style="width:627px;">For live cell imaging. Dishes such as the MatTek allow microscopic visualization at 600X or 1000X magnification through use of an inverted epifluorescent or confocal microscope.&#160; These specific dishes are 3.5 cm nominal diameter, 3.3 cm inside diameter, with 20 mm diameter #1.5 thickness cover slips inserted into the bottoms.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Photoshop CS6</td>
			<td style="width:248px;">Adobe</td>
			<td style="width:161px;"></td>
			<td style="width:627px;">Adobe photoshop or similar programs can be used to pseudocolor and merge light microscopic and fluorescent images.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Mathematica 10</td>
			<td style="width:248px;">Wolfam</td>
			<td style="width:161px;"></td>
			<td style="width:627px;">For generation of two-dimensioonal isocontour isobolograms and three-dimensional surface isobolograms.</td>
		</tr>
	</tbody>
</table>

high throughput, real-time, dual-readout testing of intracellular antimicrobial activity and eukaryotic cell cytotoxicity

세포 내 항균 활성 및 진핵 세포의 세포 독성의 전통적인 조치는 엔드 포인트 분석에 의존하고 있습니다. 이러한 엔드 포인트 분석은 세포 용해, 집락 형성 단위 결정, 또는 시약을 추가로 판독을하기 전에 몇 가지 추가 실험 단계를 필요로한다. 예를 들어, 높은 처리량 스크리닝 분석법 중에 수천을 수행 할 때, 이러한 유형의 분석법에 필요한 하류 노력은 상당하다. 따라서, 높은 처리량 항균 검색을 용이하게하기 위해, 우리는 동시에 세포 내 세균 증식의 억제제를 식별하고, 진핵 세포의 세포 독성을 평가하기위한 실시간 분석을 개발 하였다. 특히, 실시간으로 세포 내 세균의 성장 검출은 세균 룩스 오페론 (1 차 세대 분석) 또는 형광 단백질 기자 (2 차 생성, 직교 분석) 중 하나와 세균 검사 균주를 표시하여 사용 하였다. 비 독성, 세포 막 impermeant 핵산 결합 염료또한 대 식세포의 초기 감염시 추가되었습니다. 이 염료는 가능한 세포에서 제외됩니다. 그러나, 비 - 실행 가능한 숙주 세포 엔트리 핵 DNA의 형광 표식 (디옥시리보 핵산)를 허용 막 완전성을 잃는다. 특히, DNA는 숙주 세포 사멸의 용액 기반의 판독을 제공하고, 형광 양자 수율이 크게 증가와 관련된 바인딩. 우리는 마이크로 플레이트 포맷에서 높은 처리량 스크린을 수행하고, 현미경으로 세포의 성장과 세포 독성을 평가하기 위해 결합 분석을 사용 하였다. 특히 항균제 함께 적용 둘 이상의 항균제의 결합 된 효과는 개별적으로 적용될 때보다 큰 시너지 효과를 입증 할 수있다. 다른 농도에서 항생제의 조합 순열을 평가해야하기 때문에 세포 내 병원균에 대한 시험 관내 시너지 효과를 테스트하는 것은 일반적으로 엄청난 작업입니다. 그러나, 우리는 우리의 실시간 분석이 자동화, 디지털 분배 기술 쪽과 함께 발견손쉬운 시너지 테스트 ermitted. 이러한 접근 방식을 사용하여, 우리는 체계적으로 세포 내 병원균, 레지오넬라 뉴모 필라에 혼자 항생제의 많은 수의 행동과 조합을 조사 할 수 있었다.

마이크로 세포 성장 분석 도 1은 분석 단계도이다. 도시 자동화 단계가 수동으로 수행 될 수있다. 그러나, 처리량이 큰 액체 핸들링 시스템을 이용하여 용이하게된다. 도 2는 384- 웰 마이크로 플레이트, 듀얼 판독 실시간 세포 성장 및 (Lp02)은 중 어느 하나...

Watch this Scientific Journal Video about High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity at JoVE.com

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

높은 처리량, 실시간, 세포 내 항균 활동의 이중 판독 테스트 및 진핵 세포의 세포 독성

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

The overall goal of this protocol is to use a single combination real-time assay to identify specific small molecule inhibitors of intracellular bacterial growth that are non-toxic to host cells. This method can help answer key questions in the antimicrobial discovery field. Specifically, what types of compounds can penetrate into intracellular compartments and kill pathogens without harming the host cell?The main advantages of this technique are the bacterial growth mammalian cell toxicity are detected simultaneously using non-destructive, real-time assays. Generally, individuals new to this method will struggle with the scalability that it affords. Assaying greater than ten thousand compounds in a session requires a whole new set of techniques and equipment.In addition to Lucius, demonstrating a communal commitment to academic antimicrobial discovery and translational diagnostic microbiology are post-doctoral fellows, KP Smith, Yoon-Suk Kang, Jennifer Tsang, Thea Brennan-Krohn, and undergraduate Kat Truelson. To prepare host cells, culture J774A. 1 macrophage is in suspension in RPMI 1640, with nine percent iron supplemented calf serum.Initially passage cells in tissue culture flasks. After cells have become confluent in a 75 square centimeter tissue culture flask in 15 milliliters of medium, split the cells by scraping and diluting them to 65 milliliters with the same type of medium. Return 15 milliliters to the tissue culture flask, and transfer 50 milliliters to a 250 milliliter bacterial shaker flask.Aerate by setting rotation speed at approximately 120 revolutions per minute. For consistent growth, incubate at exactly five percent carbon dioxide and 37 degrees Celsius. Harvest the cells when they reach a density in the range of two point five million cells per milliliter, to five million cells per milliliter.Ensure dead cell percentage does not exceed 25 percent, as dead cells will increase background noise in cytotoxicity assays. It's critical to prevent cell settling in reservoirs when you're dispensing large volumes of macrophages and bacteria with liquid handlers. Swirl reservoirs every few minutes while dispensing to avoid non-uniformity and assay plate wells.Plate the cells in white tissue culture treated, 384 well microplates at 50, 000 cells in 30 microliters of tissue culture medium per well. Incubate microplates overnight to achieve ninety percent confluence on the day of the experiment. Prepare patches of bacteria for experiments, by spreading organisms thickly on a new BCYE plate and incubating one day to obtain confluent growth.Re-suspend the organisms in the same tissue culture medium used for the J774A. 1 cells. To perform the macrophage infection, add test compounds of interest, including screening compounds, and positive and negative controls for bacterial growth inhibition in eukaryotic cell lysis.Stock solutions should be dissolved at greater than or equal to 500x in DMSO or an aqueous solution, to allow sufficient dilution of the vehicle. Dilute luminescent bacteria to a target of two point five million CFU per millileter in tissue culture medium. Then, add appropriate non-toxic membrane impermeant nucleic acid finding dye at two point five x final assay concentration.Add 20 microliters of this mixture to each culture well. The final assay volume is 50 microliters and the final bacterial concentration should be one million CFU per milliliter for the lux epron reporter, or four million CFU per milliliter for the fluorescent protein reporter. Incubate at 37 degrees Celsius, for one to three days in five percent carbon dioxide at 100 percent relative humidity to prevent evaporative edge effects.To avoid temperature associated edge effects when reading assay results, thermally equilibrilate microplates prior to luminescence reading by placement in a single layer on a lab bench with lids ajar for approximately 20 minutes. Read bacterial growth and eukaryotic cell toxicity on a microplate luminometer and flourometer as appropriate for the reporters being used. Return microplates to the incubator if real-time readout at later time points is desired.Analyze the data as described in the text protocol. For dose response and synergy testing experiments, prepare macrophages in bacteria as before. Just prior to macrophage infection, or axenic incubation, use an automated liquid handling system to facilitate single dose response in combinatorial synergy testing set up through direct automated addition of antimicrobial dilutions according to manufacturers protocol.Add antimicrobials of experimental interest in a serial doubling dilution series. The goal is to span, on the high end, a concentration that completely eliminates growth, and on the low end, a concentration that shows no obvious activity. Shown here, are representative results over time for bacterial luminescence and cytotoxicity associated fluorescence.Note the contrasting effects of antimicrobial treatment and eukaryotic cell cytotoxic detergent. Dual IC50 and CC50 dose response determination in the same screening wells was used to determine selectivity for the antibiotic Doxycycline. Bacterial replication was inhibited by intermediate concentrations of antibiotic.However, cytotoxicity was observed at high concentrations consistent with the selectivity of approximately 100. The same assay format was used to test two dimensional dose response for combinations of Minocycline and Azithromycin. Isocontours connect points of equal intracellular growth inhibition.Here, concave isocontours suggested strong synergistic effects for the two drugs. Digital dispensing robotics for assay setup real-time readout, and high-throughput format enabled combinatorial triple synergy testing. Here, observation of a distinctly concave surface suggests a high degree of synergistic effect for combinations of Minocycline, Azithromycin, and Rifampin against intracellular growth of legionella pheumophila.After watching this video, you should have a good understanding of how to combine either fluorescent or luminescent assay read-out with real-time, cytotoxicity testing in high-throughput assay format. Following this procedure, other methods like confocal microscope can be applied answer questions about biological event in the whole cell, which are also sited with their antimicrobe activity or whole cell cytotoxicity. Though we studied the effects on legionella replication, the same techniques can also be applied to other intracellular pathogens such as Brucella, and microbacteria.We first conceived to this protocol when trying to figure out an assay simple and reliable enough to use in a very high-throughput screening format. Don't forget that working with bacterial pathogens, cell lines, and robotic equipment has some inherent risks and appropriate precautions should be taken, such as use of appropriate personal protective equipment while performing this procedure.

Research

JoVE Journal

Immunology and Infection

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &#946;-arrestin

Leukotriene B의 실시간 영상 4 중재 셀 마이 그 레이션 및 β - arrestin로 BLT1 상호 작용

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Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

T 세포 양적 높은 처리량 단일 세포 세포 독성 분석

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Real-time Live Imaging of T-cell Signaling Complex Formation

T 세포의 실시간 라이브 영상은 복잡한 형성을 신호

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

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

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 ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

호중구 윤회에서 생리 흐름 중 내피 세포 - 세포 접합의 실시간 영상

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

높은 처리량 정량 실시간 RT-PCR 분석 유형 I과 III 인터페론 하위 유형의 발현 양상을 결정하는

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

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

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

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 ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

인간 NK 세포 활동의 평가 대 한 흐름 Cytometry 기반 세포 독성 분석 실험

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

고밀도 지단백에 지질 과산화의 높은 처리량 측정을 위한 생 화 확 적인 세포-무료 Fluorometric 효소 분석 결과

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

루시퍼라제 기반 성장 분석을 사용하여 세포내 톡소플라즈마 곤디성장에 대한 화학적 억제제 효율 결정

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...