Research reported in this manuscript was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R01AI099122 to J.E.K. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We would like to thank Jennifer Smith, David Wrobel, Su Chiang, Doug Flood, Sean Johnston, Jennifer Nale, Stewart Rudnicki, Paul Yan, Richard Siu, and Rachel Warden from the ICCB-Longwood Screening Facility and/or the National Screening Laboratory for the New England Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases (supported by U54AI057159) for their assistance in development and performance of high-throughput screening assays. We also would like to thank Kenneth P. Smith for helpful comments on the manuscript.

1. Real Time Intracellular Growth and Eukaryotic Cell Cytotoxicity Assay
<ol>
	<li>Preparing Host Cells (J774A.1 Cells)
	<ol>
		<li>Culture J774A.1 Mus musculus macrophage-like cells in suspension in RPMI 1640 with 9% iron-supplemented calf serum. Initially passage in tissue culture flasks. After cells have become confluent in a 75 cm2 tissue culture flask in 15 ml of medium, split by scraping and dilution to 65 ml with the same type of medium, of which 15 ml is returned to the tissue culture flask...

<ol>
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C., Lorian, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+9.">Ch. 9.</a> Antimicrobial combinations. In: Antibiotics in Laboratory Medicin. , 330-396 (1996).</li><li>Jones, R. E., Zheng, W., McKew, J. C., Chen, C. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+direct+compound+dispensing+method+using+the+HP+D300+digital+dispenser.">An alternative direct compound dispensing method using the HP D300 digital dispenser.</a> J Lab Autom. 18 (5), 367-374 (2013).</li><li>Odds, F. 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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. 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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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We describe real-time assays for simultaneous detection of intracellular bacterial growth and host cell cytotoxicity. There are several critical steps in the protocol. First, for robust assay performance, there must be sufficient spectral separation between bacterial and cytotoxicity readouts. Such separation is intrinsic for combinations of luciferase operon reporters and fluorescent DNA-binding dyes. However, based on our experience (Table 1-3, Figure 2), use of dual, fluorescent reado...

Pathogens that grow or reside temporarily in intracellular compartments are difficult to therapeutically eradicate. Obligate or relatively obligate intracellular pathogens such as Legionella pneumophila, Coxiella burnetii, Brucella spp., Francisella tularensis, and Mycobacterium spp. often require prolonged courses of antimicrobial therapy for cure that may range from months to even years. Furthermore, extracellular pathogens may transiently occupy intracellular niches and in this way escape clearance by normal courses of antimicrobial therapy and later emerge to start new rounds of virulent infection. Staphylococcus aureus1 and uropathogenic Enterobacteriaceae2,3 infections are two increasingly recognized examples. Therefore, a fundamental drug discovery goal is to identify novel antimicrobials that penetrate into intracellular compartments. Optimal therapy to quickly eradicate intracellular organisms and prevent development of resistance through sub-inhibitory antimicrobial exposure is especially desirable.
To this end, we developed a high-throughput screening technology to identify intracellular-penetrant antimicrobials targeting the intracellular growth of the model pathogen, Legionella pneumophila.4 Previous clinical observations indicate that standard antimicrobial susceptibility testing did not accurately predict in vivo therapeutic efficacy against this organism.5 Specifically, this was because major classes of antimicrobials such as &#946;-lactams and aminoglycosides, although highly effective against axenically grown Legionella, do not sufficiently penetrate into the intracellular compartments where Legionella resides.5,6 Later evidence suggested that technically more complex intracellular growth assays effectively predicted clinical efficacy.7 Unfortunately, these assays were extremely laborious endpoints assays, requiring infected macrophages, treated with antimicrobials, to be lysed at different times points for colony forming unit enumeration. Such assays are impractical to do on a large scale and are unsuitable for high-throughput drug discovery.
Therefore, we developed technology for real-time determination of intracellular bacterial growth.6 This was accomplished through use of a bacterial strain modified through integration of either a bacterial luciferase operon8 (first generation assay, described previously)4 or fluorescent protein9 reporters (second generation, orthogonal assay, described here) into the bacterial chromosome. In this way, luminescent or fluorescent signal provides a surrogate, real-time readout of bacterial number.
However, these attributes do not address a major confounder in intracellular infection assays, off-target effects on host cells. In particular, the death of the host cell inherently limits intracellular growth and leads to false positive identification of antimicrobial effect. As many compounds in screening libraries are eukaryotic cell toxic, such false positives would overwhelm true antimicrobials, necessitating a large number of follow-up, endpoint cytotoxicity assays for resolution.
Thus, it was of great interest to be able to assess eukaryotic cell viability and intracellular growth simultaneously. Notably, a characteristic of non-viable eukaryotic cells is loss of cell membrane integrity. Probes that test the permeability of the cell membrane may therefore be used to assess cell viability. We previously characterized the ability of a series of putatively cell membrane-impermeant, fluorescent, DNA-binding dyes to access and stain nuclear DNA of dead cells.4 On binding nuclear DNA, these dyes display a large increase in quantum fluorescent yield resulting in increased signal over background solution fluorescence. As such, these dyes provided a quantitative readout of eukaryotic cell death.4 Notably, we found that several were non-toxic themselves during prolonged co-incubation with J774 macrophages. When added during initial infection, they provided a real-time, fluorescent readout of eukaryotic cell death that can be measured by a microplate fluorimeter or observed microscopically.
Therefore, by combining use of a bacterial reporter and non-toxic, membrane-impermeant, DNA-binding dyes, we were able to develop a simple, non-destructive, real-time assay to measure both bacterial load and eukaryotic cell cytotoxicity simultaneously. This assay has allowed us to screen in 384-well plate format ~10,000 known bioactives including ~250 antimicrobials and &#62;240,000 small molecules with functionally uncharacterized activity for the ability to inhibit intracellular growth of Legionella pneumophila, while at the same time generating eukaryotic cell cytotoxicity data for each compound.6 Our analysis of known antimicrobials against intracellular growth of Legionella was the most comprehensive exploration of this type to date.6
Based on the efficiency of our assay format, we also subsequently explored the potentially synergistic effects of known antimicrobials when used in combination. One of the most common synergy tests, the so-called checkerboard assay, is standardly performed by assessing combinatorial effects of two-fold serial dilutions of two or more antimicrobials.10 In these assays, synergy is defined by the observation of greater effect when two or more antimicrobials are applied together than the sum of the effects of each applied separately. Of note, heretofore, only focused and selective synergy testing was performed against intracellular Legionella pneumophila because of the great effort involved in traditional endpoint assays multiplied by the combinatorial permutations required.
To facilitate synergy testing, we made use of our real-time intracellular growth/eukaryotic cytotoxicity assay in combination with automated digital dispensing technology6. This automation permitted us to dispense serial dilutions of compounds dissolved in DMSO or aqueous solution alone or in combination in 384-well format.11 Furthermore, such robust liquid handling technology permitted us to easily perform higher resolution, square-root-of-two (rather than the standard, lower resolution, doubling) dilution combinations to achieve higher levels of specificity in our two-dimensional, checkerboard synergy analysis. This resolution was especially valuable in addressing concerns in the synergy field about reproducibility when using two-fold dilution series12. Lastly, our assay was quantitative and also therefore measured gradations of inhibition. As a result, the assay captured the entirety of inhibitory information, expressible in isocontour isobolograms in which isocontours connect combinatorial concentrations with similar levels of growth inhibition.6 This plotting strategy allowed visualization of combinatorial dose-response curves. To illustrate our methodology, we describe our protocol for performing these assays and show representative results.

<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

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

Microplate intracellular growth assay
Figure 1 diagrams the assay steps. The automated steps shown can be performed manually. However, throughput is greatly facilitated using liquid handling systems.
Figure 2 shows representative results from a 384-well microplate, dual-readout, real-time intracellular growth and eukaryotic cell cytoto...

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.

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.

high-throughput-real-time-dual-readout-testing-intracellular

Research

JoVE Journal

Immunology and Infection

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

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

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

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

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

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

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

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

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

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

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

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

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

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 locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

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

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.

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

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

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

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

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

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

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

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

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