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

Research rapporterade i detta manuskript stöddes av National Institute of Allergy och infektionssjukdomar av National Institutes of Health i tilldelning nummer R01AI099122 att JEK Innehållet är ensamt ansvarig för författare och inte nödvändigtvis representerar officiella ståndpunkter National Institutes of Hälsa. Vi vill tacka Jennifer Smith, David Wrobel, Su Chiang, Doug Flood, Sean Johnston, Jennifer Nale, Stewart Rudnicki, Paul Yan, Richard Siu, och Rachel Warden från ICCB-Longwood Screening Facility och / eller National Screening laboratoriet för New England regionala kompetenscentrum i Biodefense och Emerging Infectious Diseases (stöds av U54AI057159) för deras hjälp i utvecklingen och resultatet av high-throughput screening-analyser. Vi vill också tacka Kenneth P. Smith hjälp kommentarer på manuskriptet.

1. Real Time Intracellulär tillväxt och eukaryot cell cytotoxicitet <ol><li> Förbereder värdceller (J774A.1 Cells) <ol><li> Kultur J774A.1 Mus musculus makrofagliknande celler i suspension i RPMI 1640 med 9% järn-kompletterat kalvserum. Inledningsvis passage i vävnadsodlingskolvar. Efter det att cellerna har blivit sammanflytande i en 75 cm 2 vävnadsodlingskolv på 15 ml medium, uppdelad genom skrapning och utspädning till 65 ml med samma typ av medium, varav 15 ml återförs till vävnadso...

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J., 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=Improving+FRET+dynamic+range+with+bright+green+and+red+fluorescent+proteins.">Improving FRET dynamic range with bright green and red fluorescent proteins.</a> Nat Methods. 9 (10), 1005-1012 (2012).</li><li>Berger, K. H., Merriam, J. J., Isberg, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+intracellular+targeting+properties+associated+with+mutations+in+the+Legionella+pneumophila+dotA+gene.">Altered intracellular targeting properties associated with mutations in the Legionella pneumophila dotA gene.</a> Mol Microbiol. 14 (4), 809-822 (1994).</li><li>Berger, K. H., Isberg, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+defects+in+intracellular+growth+complemented+by+a+single+genetic+locus+in+Legionella+pneumophila.">Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila.</a> Mol Microbiol. 7 (1), 7-19 (1993).</li><li>Vogel, J. P., Isberg, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+of+Legionella+pneumophila.">Cell biology of Legionella pneumophila.</a> Curr Opin Microbiol. 2 (1), 30-34 (1999).</li><li>Kirby, J. E., Isberg, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legionnaires'+disease:+the+pore+macrophage+and+the+legion+of+terror+within.">Legionnaires' disease: the pore macrophage and the legion of terror within.</a> Trends Microbiol. 6 (7), 256-258 (1998).</li><li>Kirby, J. E., Vogel, J. P., Andrews, H. L., Isberg, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+pore-forming+ability+by+Legionella+pneumophila.">Evidence for pore-forming ability by Legionella pneumophila.</a> Mol Microbiol. 27 (2), 323-336 (1998).</li><li>Vogel, J. P., Andrews, H. L., Wong, S. K., Isberg, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugative+transfer+by+the+virulence+system+of+Legionella+pneumophila.">Conjugative transfer by the virulence system of Legionella pneumophila.</a> Science. 279 (5352), 873-876 (1998).</li><li>Song, H., Fares, M., Maguire, K. R., Sid&#233;n, A., Pot&#225;cov&#225;, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+Effects+of+Tetracycline+Analogues+(Doxycycline,+Minocycline+and+COL-3)+in+Acute+Myeloid+Leukemia+HL-60+Cells.">Cytotoxic Effects of Tetracycline Analogues (Doxycycline, Minocycline and COL-3) in Acute Myeloid Leukemia HL-60 Cells.</a> PLoS ONE. 9 (12), e114457 (2014).</li><li>Isberg, R. R., Falkow, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+genetic+locus+encoded+by+Yersinia+pseudotuberculosis+permits+invasion+of+cultured+animal+cells+by+Escherichia+coli+K-12.">A single genetic locus encoded by Yersinia pseudotuberculosis permits invasion of cultured animal cells by Escherichia coli K-12.</a> Nature. 317 (6034), 262-264 (1985).</li><li>Niles, A. L., Moravec, R. A., Riss, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+in+vitro+cytotoxicity+assays+for+drug+development.">Update on in vitro cytotoxicity assays for drug development.</a> Expert Opin Drug Discov. 3 (6), 655-669 (2008).</li><li>Niles, A. L., Moravec, R. A., Riss, T. 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Vi beskriver realtids analyser för samtidig detektion av intracellulära bakterietillväxt och värdcell cytotoxicitet. Det finns flera viktiga steg i protokollet. Först för robust analysprestanda, måste det finnas tillräckligt spektral separation mellan bakterie- och cytotoxicitet avläsning. En sådan separation är inneboende för kombinationer av luciferas Operon reportrar och fluorescerande DNA-bindande färgämnen. Baserat på vår erfarenhet (Tabell 1-3, Figur 2), användnin...

Patogener som växer eller uppe tillfälligt i intracellulära fack är svåra att terapeutiskt utrota. Förpliktigar eller förpliktigar relativt intracellulära patogener såsom Legionella pneumophila, Coxiella burnetii, Brucella spp. , Francisella tularensis, och Mycobacterium spp. ofta kräver långvarig kurser antimikrobiell behandling för botemedel som kan variera från månader till och med år. Dessutom kan extracellulära patogener gående ockupera intracellulära nischer och på så sätt undgå clearance av normala kurser antimikrobiell behandling och senare dyker starta nya omgångar av virulent infektion. Staphylococcus aureus 1 och uropatogena Enterobacteriaceae 2,3 infektioner är två alltmer erkända exempel. Därför är en grundläggande läkemedelsutveckling mål att identifiera nya antimikrobiella medel som tränger in i intracellulära fack. Optimal terapi för att snabbt utrotaintracellulära organismer och förhindra utveckling av resistens genom sub-hämmande antimikrobiella exponering är särskilt önskvärd. För detta ändamål har vi utvecklat en high-throughput screening teknik för att identifiera intracellulära-penetrerande antimikrobiella medel som riktar sig till intracellulära tillväxten av modellen patogen, Legionella pneumophila. 4 Tidigare kliniska observationer tyder på att standarden resistensbestämning inte exakt hade förutsäga in vivo terapeutisk effekt mot denna organism. 5 Närmare bestämt var detta eftersom stora klasser av antimikrobiella medel såsom p-laktamer och aminoglykosider, men mycket effektiv mot axeniskt odlade Legionella, inte tillräckligt tränga in i intracellulära avdelningar där Legionella är bosatt. 5,6 Senare bevis föreslog att tekniskt mer komplexa intracellulära tillväxtanalyser effektivt förutspått klinisk effekt. 7 </sup&gt; Tyvärr är dessa analyser var extremt mödosamma endpoints analyser, kräver infekterade makrofager, som behandlats med antimikrobiella medel, som skall lyseras vid olika tidpunkter poäng för kolonibildande enhet uppräkning. Sådana analyser är opraktiskt att göra i stor skala och är olämpliga för high-throughput drug discovery. Därför har vi utvecklat teknologi för realtids-bestämning av intracellulär bakterietillväxt. 6 Detta åstadkoms genom användning av en bakteriestam modifierad genom integration av antingen en bakteriell luciferas operon 8 (första generationen analysen tidigare beskrivits) 4 eller fluorescerande protein 9 reportrar (andra generationens, ortogonala analys, beskrivs här) i bakteriekromosomen. På detta sätt, självlysande eller fluorescerande signal ger ett surrogat realtid avläsning av bakteriell nummer. Men dessa attribut inte upp en stor confounder i intracellulär infection analyser, off-target effekter på värdceller. I synnerhet död av värdcellen i sig begränsar intracellulär tillväxt och leder till falsk positiv identifiering av antimikrobiell effekt. Som många föreningar i screeningsbibliotek är eukaryota celler giftiga sådana falska positiva skulle överväldiga verkliga antimikrobiella medel, vilket kräver ett stort antal uppföljnings endpoint cytotoxicitetsanalyser för upplösning. Således var det av stort intresse att kunna bedöma eukaryot cellviabilitet och intracellulär tillväxt samtidigt. Noterbart är en egenskap av icke-viabla eukaryota celler förlust av cellmembranintegritet. Prober som testar permeabiliteten av cellmembranet kan därför användas för att bedöma cellviabilitet. Vi tidigare karaktäriserat förmågan hos en serie av förmodat cellmembran impermeant, fluorescerande, DNA-bindande färgämnen för att få tillgång till och ge fläckar kärn-DNA av döda celler. 4 Den bindande nukleärt DNA, dessa färgämnen visar en stor ökning av quantum fluorescerande utbyte resulterar i ökad signal över bakgrunds lösning fluorescens. Som sådana dessa färgämnen förutsatt en kvantitativ avläsning av eukaryot celldöd. 4 Särskilt fann vi att flera var giftfri sig under långvarig samtidig inkubation med J774 makrofager. När läggas under den första infektionen, de tillhandahöll en realtid, fluorescerande avläsning av eukaryot celldöd som kan mätas genom en mikro fluorimeter eller observeras mikroskopiskt. Därför genom att kombinera användning av en bakteriell reporter och giftfri, membran ogenomtränglig, DNA-bindande färgämnen, kunde vi utveckla en enkel, icke-förstörande, realtidsanalys för att mäta både bakteriehalten och eukaryota celler cytotoxicitet samtidigt. Denna analys har gjort det möjligt för oss att screena i 384-brunnar format ~ 10000 kända bioaktiva substanser inklusive ~ 250 antimikrobiella och&gt; 240.000 små molekyler med funktionellt karaktäriserad aktivitet för förmågan att hämma intracellulär tillväxtLegionella pneumophila, medan på samma gång generera eukaryota cell cytotoxicitet data för varje förening. 6 Vår analys av kända antimikrobiella medel mot intracellulär tillväxt av Legionella var den mest omfattande undersökning av denna typ hittills. 6 Baserat på effektiviteten i vår analysformat, vi senare undersökte också de potentiellt synergistiska effekterna av kända antimikrobiella medel när de används i kombination. En av de vanligaste synergitester, den så kallade checkerboard-analysen, är standardmässigt utförs genom att bedöma kombinatoriska effekter av två-faldiga seriespädningar av två eller flera antimikrobiella medel. 10 I dessa analyser är synergi definieras av observationen av större effekt när två eller flera antimikrobiella medel används tillsammans än summan av effekterna av varje tillämpas separat. Notera hittills bara fokuserat och selektiv samverkan test utfördes mot intracellulära Legionella pneumophila </em&gt; på grund av den stora insats som krävs i traditionella endpoint analyser multiplicerat med kombi permutationer krävs. För att underlätta samverkan testning, gjorde vi användning av vår realtids intracellulär tillväxt / eukaryot cytotoxicitetsanalys i kombination med automatiserad digital dispenseringsteknik 6. Denna automatisering möjligt för oss att dispensera seriespädningar av föreningar lösta i DMSO eller vattenlösning ensamma eller i kombination i 384-brunnsformat. 11 Vidare medgivit en sådan robust flytande teknik hantering oss att enkelt utföra högre upplösning, kvadratroten av två-(snarare än den vanliga, lägre upplösning, en fördubbling) utspädnings kombinationer för att uppnå högre nivåer av specificitet i vår tvådimensionella, schack synergi analys. Resolutionen var särskilt värdefullt att ta itu med problem i samverkan fältet om reproducerbarhet när tvåfaldig spädningsserie 12. Slutligen vår analys var kvantitativt och även finsöre mätt graderingar av inhibition. Som ett resultat, analysen fångade helheten av inhiberande informationen, som kan uttryckas i isokontur isobolograms där isocontours ansluta kombinatoriska koncentrationer med liknande nivåer av tillväxthämning. 6 Denna plottning strategi tillät visualisering av kombi dos-responskurvor. För att illustrera vår metodik, beskriver vi våra protokoll för att utföra dessa analyser och visar representativa resultat.

<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

Traditionella mått på intracellulär antimikrobiell aktivitet och eukaryot cell cytotoxicitet lita på endpoint analyser. Sådana endpoint analyser kräver flera ytterligare experimentella steg före avläsning, såsom cell-lys, kolonibildande enhet bestämning, eller tillsats av reagens. Vid utförande av tusentals analyser, till exempel under high-throughput screening, är den nedströms belägna ansträngning som krävs för dessa typer av analyser betydande. Därför att underlätta hög genomströmning antimikrobiell upptäckt, har vi utvecklat en realtidsanalys för att samtidigt identifiera hämmare av intracellulära bakterietillväxt och bedöma eukaryot cell cytotoxicitet. Specifikt realtid intracellulär bakterietillväxt detektering aktiveras genom att markera bakterie screening stammar med antingen en bakteriell lux-operonet (1 st generationen analys) eller fluorescerande protein reportrar (2: a generationens, ortogonala analys). En icke-toxisk, cellmembran impermeant, nukleinsyrabindande färgämnetillsattes också vid initial infektion av makrofager. Dessa färgämnen är uteslutna från levande celler. Men icke livsdugliga värdceller förlorar membranintegritet medger inträde och fluorescerande märkning av kärn-DNA (deoxiribonukleinsyra). Notably, DNA-bindning är förknippad med en stor ökning av fluorescerande kvantutbyte som ger en lösningsbaserad utläsning av värd celldöd. Vi har använt denna kombinerade analys för att utföra en hög genomströmning skärm i mikroformat, och för att bedöma intracellulära tillväxt och cytotoxicitet av mikroskopi. Anmärkningsvärt, kan antimikrobiella visar synergi där den kombinerade effekten av två eller flera antimikrobiella medel när de appliceras tillsammans är större än när det appliceras separat. Testning för in vitro synergi mot intracellulära patogener är normalt en ofantlig uppgift som kombinatoriska varianter av antibiotika vid olika koncentrationer skall bedömas. Vi fann emellertid att vår realtidsanalys i kombination med automatiserad, digital dispenseringsteknik permitted facile synergi testning. Med hjälp av dessa metoder, kunde vi systematiskt kartlägga inverkan av ett stort antal enbart antimikrobiella medel och i kombination mot den intracellulära patogenen, Legionella pneumophila.

Mikroplattan intracellulära tillväxtanalys Figur 1 visar i diagramanalyssteg. De automatiserade stegen som visas kan utföras manuellt. Dock är genomströmningen underlättas avsevärt med hjälp av system vätskehantering. Figur 2 visar representativa resultat från en 384-brunnars mikroplatta, dual-avläsning i realtid intracel...

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.

Hög genomströmning, i realtid, med dubbla avläsning Provning av intracellulära antimikrobiell aktivitet och eukaryot cell cytotoxicitet

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

Realtid avbildning av Leukotriene B 4 Medierad Cell Migration och BLT1 Interaktioner med β-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

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

Kvantitativ hög kapacitet encelliga cytotoxicitetsanalys för T-celler

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

Real-time Live avbildning av T-cellsignalering Complex Formation

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

En mikroskopisk fenotypisk analys för kvantifiering av intracellulär mykobakteri anpassad för screening med högt genomströmning/högt innehåll

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

Realtid avbildning av endotelceller-cells Junctions Under neutrofila Trans under fysiologiska Flow

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

Hög-throughput kvantitativ realtids-RT-PCR-analys för bestämning av uttryck Profiler av typ I och III Interferon Subtyper

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

System för Efficacy och cytotoxicitet Screening av inhibitorer Inriktning Intracellulär<em&gt; Mycobacterium tuberculosis</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

Ett flöde flödescytometri-baserade cytotoxicitet analys för bedömning av människans NK cellsaktivitet

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

Cell-free biokemiska Fluorometric enzymatisk analys för High-throughput mätning av lipidperoxidation i hög Density Lipoprotein

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

Bestämning av kemisk hämmare effektivitet mot intracellulära Toxoplasma Gondii tillväxt med hjälp av en Luciferase-baserad tillväxt analys

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