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 and 50 ml is transferred to a 250 ml bacterial shaker flask.</li>
		<li>For scale-up, culture in suspension in 250 ml and/or 1,000 ml bacterial flasks filled to one fifth volume with tissue culture medium. Aerate by setting rotation speed at approximately 120 revolutions per minute. For consistent growth, incubate at exactly 5% CO2 and 37 &#176;C.</li>
		<li>Harvest cells when they reach density in the range 2.5 x 106 cells/ml to 5 x 106 cells/ml. Ensure dead cell percentage (most easily assayed by trypan blue staining) does not exceed 25%, as dead cells will increase background noise in cytotoxicity assays.</li>
		<li>Plate in white, tissue culture-treated, 384-well microplates at 5 x 104 J774A.1 cells in 30 &#181;l tissue culture medium per well. Incubate microplates overnight to achieve 90% confluence on day of experiment.</li>
	</ol>
	</li>
	<li>Preparing Luminescent or Fluorescent Legionella pneumophila
	<ol>
		<li>Passage L. pneumophila (Lp02::flaA::lux8) bacteria on appropriate medium, i.e., buffered charcoal yeast extract (BCYE) medium. If using a thymidine auxotrophic strain, supplement medium with 100 &#181;g/ml thymidine. Prepare patches of bacteria for experiments by spreading organisms thickly on a new BCYE plate and incubating one day (if from a previous plate passage) or two to three days (if from frozen stock) to obtain confluent growth.
		<ol>
			<li>Prepare BCYE plates by dissolving 10 g yeast extract, 10 g N-(2-acetamido)-2-aminoethanesulfonic acid, 0.35 g K2HPO4, and 1 g monobasic potassium &#945;-ketoglutarate in 950 ml deionized water. Adjust to pH 6.9 with Potassium hydroxide (&#8805;2.5 ml of 11.9 molar solution).</li>
			<li>Add 15 g agar and 2 g activated charcoal; and bring to 1 L final volume. Add a magnetic stir bar and autoclave. Cool medium to 55 &#176;C and add filter-sterilized solutions containing 0.4 g L-cysteine and 0.42 g Ammonium iron(III) citrate dissolved in deionized water. Use magnet stir plate to mix prior to pouring Petri plates.</li>
			<li>For testing of L. pneumophila growth in axenic growth medium, prepare ACES-yeast extract broth (AYE) by combining all chemical reagents used for BCYE except for charcoal and agar. Filter sterilize and use immediately, or freeze for later experiments.</li>
		</ol>
		</li>
		<li>Resuspend organisms in the same tissue culture medium used for J774A.1 cells with 100 &#181;g/ml thymidine supplementation as appropriate.</li>
	</ol>
	</li>
	<li>Macrophage Infection
	<ol>
		<li>Add test compounds of interest (screening compounds, and positive and negative controls for bacterial growth inhibition and eukaryotic cell lysis). Preferably, dissolve stock solutions at &#8805;500x in DMSO (dimethyl sulfoxide) or an aqueous solution to allow sufficient dilution of vehicle.
		<ol>
			<li>Although stock solutions can be stored frozen, avoid freeze-thaw cycles for labile compounds and antimicrobials.</li>
		</ol>
		</li>
		<li>Dilute luminescent bacteria to target of 2.5 x 106 CFU (colony forming unit)/m and fluorescent reporter-labeled bacteria to 1.0 x 107 CFU/ml in tissue culture medium and add appropriate non-toxic, membrane-impermeant, nucleic acid-binding dye at 2.5x final assay concentration.</li>
		<li>Add 20 &#181;l of mixture to each J774A.1 culture well, final assay volume 50 &#181;l, final bacterial concentration 1 x 106 CFU/ml (lux operon reporter) or 4 x 10 106 CFU/ml (fluorescent protein reporter).</li>
		<li>Incubate at 37 &#176;C for 1-3 days in 5% CO2 at 100% relative humidity to prevent evaporative edge effects.</li>
	</ol>
	</li>
	<li>Assay Readout
	<ol>
		<li>Read bacterial growth and eukaryotic cell toxicity on a microplate luminometer and fluorimeter as appropriate for reporters being used.
		<ol>
			<li>To avoid temperature-associated edge effects, thermally equilibrate microplates prior to luminescence reading by placement in a single layer on a lab bench with lids ajar for approximately 20 min (or in a biological safety cabinet with lids off for approximately 10 min).</li>
		</ol>
		</li>
		<li>Return microplates to incubator if real-time readout at later time points is desired.</li>
	</ol>
	</li>
</ol>
2. Data Analysis
<ol>
	<li>Normalize data to positive and negative controls to calculate percent or fold-inhibition for intracellular bacterial growth and percent cytotoxicity for eukaryotic cells.</li>
	<li>To assess assay robustness, calculate statistical separation between positive and negative controls for both bacterial growth inhibition and cytotoxicity using Z-factor (Z&#39;) analysis where Z&#39; = 1 - 3(&#963;p + &#963;n)/|&#181;p + &#181;n|. In this equation, &#963;p and &#963;n are the standard deviations for the positive and negative control wells, respectively; &#181;p and &#181;n are the mean values for the positive and negative control wells, respectively.
	<ol>
		<li>For high-throughput screening assays, calculate standard z-scores (or other alternative, quantitative statistical measures) to assess effects of test compounds of interest. Alternatively, calculate a simple fold-reduction or log-fold reduction as a potentially physiologically more relevant measure of effect magnitude for geometrically replicating bacteria.</li>
	</ol>
	</li>
</ol>
3. Single and Multi-dimensional (i.e., Synergy) Dose-response Testing and Data Interpretation
<ol>
	<li>Prepare macrophages and bacteria as described in protocol section 1.</li>
	<li>Just prior to infection or axenic incubation, add antimicrobials of experimental interest in a serial, doubling dilution series, with the goal of spanning on the high end a concentration that completely eliminates growth (i.e., the minimal inhibitory concentration or MIC) and on the low end a concentration that shows no obvious activity.</li>
	<li>Use an automated liquid handling system to facilitate single dose-response and combinatorial synergy testing setup through direct, automated addition of antimicrobial dilutions according to manufacturer&#39;s protocol.</li>
	<li>Consider use of sub-doubling dilutions, for example, <img alt="Equation 1" src="/files/ftp_upload/54841/54841eq1.jpg" />-fold dilution series, and assay replicates to increase accuracy and reproducibility of data.</li>
	<li>For macrophage infection, dilute luminescent bacteria to target of 2.5 x 106 CFU (colony forming unit)/ml in tissue culture medium. Add 20 &#181;l of mixture to each J774A.1 culture well, final assay volume 50 &#181;l, final bacterial concentration 1 x 106 CFU/ml; and incubate at 37 &#176;C in 5% CO2 at 100% relative humidity.
	<ol>
		<li>For axenic growth testing, dilute luminescent bacteria to final concentration of 1 x 106 CFU/ml in AYE medium, add 50 &#181;l to each well of a 384-well plate and incubate at 37 &#176;C in 5% CO2 at 100% relative humidity.</li>
	</ol>
	</li>
	<li>Calculate fractional inhibitor concentration index for antimicrobial combinations that inhibit bacterial growth. For each drug in the combination (a, b, . . n) that results in complete or &#62;99% inhibition of bacterial growth, calculate the individual fractional inhibitory concentration (FICa, b, . . n.) as follows: FICa = the concentration of compound &#34;a&#34; divided by the minimal inhibitor concentration of &#34;a&#34; by itself.. Using the same formula, calculate FICb, etc. Then calculate the combinatorial FIC index or&#160;<img alt="Equation 2" src="/files/ftp_upload/54841/54841eq2.jpg" /> FIC = FICa + FICb + . . .FICn for each inhibitory combination.
	<ol>
		<li>Use the combinatorial index that deviates furthest from 1.0 to assess synergy. Consider a cutoff of &#8804;0.5 as a conservative indication of a synergistic effect and &#8805;4, an antagonist effect.12</li>
		<li>Plot isobolograms, isocontour isobolograms, and/or isosurface isobolograms6 as alternative graphical representations of combinatorial effects.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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><tbody><tr><td>J774A.1 cells</td><td>American Type Culture Collection</td><td>TIB-67</td><td>Host cell</td></tr><tr><td>ACES</td><td>Sigma-Aldrich</td><td>A9758&amp;nbsp;</td><td>For making buffered charcoal yeast extract agar and buffered yeast extract medium</td></tr><tr><td>Yeast extract, ultrafiltered</td><td>Becton-Dickinson/Difco</td><td>210929</td><td>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><td>Alpha-ketoglutaric acid, monopotassium salt</td><td>Sigma-Aldrich</td><td>K2000</td><td>For making buffered charcoal yeast extract agar and buffered yeast extract medium</td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P5280</td><td>For making buffered charcoal yeast extract agar and buffered yeast extract medium</td></tr><tr><td>Potassium phosphate, dibasic</td><td>Thermo Fisher Scientific</td><td>P288-500</td><td>For making buffered charcoal yeast extract agar and buffered yeast extract medium</td></tr><tr><td>L-cysteine</td><td>Sigma-Aldrich</td><td>C-7755</td><td>For making buffered charcoal yeast extract agar and buffered yeast extract medium</td></tr><tr><td>Ammonium iron(III) citrate</td><td>Sigma-Aldrich</td><td>F5879</td><td>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><td>Potassium hydroxide solution, concentrated</td><td>Thermo Fisher Scientific</td><td>SP236-500</td><td>For making buffered charcoal yeast extract agar and buffered yeast extract medium</td></tr><tr><td>Deonized water</td><td>N/A</td><td>N/A</td><td>For making buffered charcoal yeast extract agar and buffered yeast extract medium</td></tr><tr><td>Thymidine (tissue culture grade)</td><td>Sigma-Aldrich</td><td>T1895</td><td>For supplementing both RPMI 1640 and buffered yeast extract agar/medium &amp;mdash; 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><td>RPMI 1640, standard formulation</td><td>Corning via Thermo Fisher Scientific</td><td>10-040-CV</td><td>For growing J774A.1 cells prior to plating; includes 2 mM L-glutamine</td></tr><tr><td>RPMI 1640 lacking phenol red</td><td>Corning via Thermo Fisher Scientific</td><td>17-105-CV</td><td>For plating J774A.1 cells in 384 well dishes (not suitable for growth prior to plating); also lacks L-glutamine &amp;mdash; supplement to 2 mM before use</td></tr><tr><td>L-glutamine, 200 mM in 0.85% NaCl (tissue culture grade)</td><td>HyClone via Thermo Fisher Scientific</td><td>SH30034.02</td><td>For supplementing RPMI 1640 lacking L-glutamine, to 2 mM final concentration</td></tr><tr><td>Iron-supplemented calf serum</td><td>Gemini Bioproducts</td><td>100-510</td><td>For supplementing RPMI 1640, to 9.1% final concentration</td></tr><tr><td>Trypan Blue solution</td><td>Sigma-Aldrich</td><td>T8154</td><td>For staining for J774A.1 cell death determination while counting cell density</td></tr><tr><td>SYTOX Green, 5 mM solution in DMSO</td><td>Thermo Fisher Scientific</td><td>S7020</td><td>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><td>Cell culture incubator</td><td>Thermo Fisher Scientific</td><td>13-255-26</td><td>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><td>Orbital shaker</td><td>BellCo Glass</td><td>7744-01010</td><td>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><td>Shaker flasks (250 ml)</td><td>ChemGlass Life Sciences</td><td>CLS-2038-04</td><td>For shaking incubation of J774A.1 cells before infection</td></tr><tr><td>Shaker clamps for flasks (250 ml)</td><td>BellCo Glass</td><td>7744-16250</td><td>For shaking incubation of J774A.1 cells before infection</td></tr><tr><td>Shaker flasks (1,000 ml)</td><td>ChemGlass Life Sciences</td><td>CLS-2038-07</td><td>For shaking incubation of J774A.1 cells before infection</td></tr><tr><td>Shaker clamps for flasks (1,000 ml)</td><td>BellCo Glass</td><td>7744-16100</td><td>For shaking incubation of J774A.1 cells before infection</td></tr><tr><td>Sponge foam caps for flasks (250 ml-1,000 ml)</td><td>ChemGlass Life Sciences</td><td>CLS-1490-038</td><td>For shaking incubation of J774A.1 cells before infection; reduces risk of contamination relative to standard metal caps</td></tr><tr><td>MultiDrop Combi programmable multichannel peristaltic pump</td><td>Thermo Fisher Scientific</td><td>5840300</td><td>For dispensing J774A.1 cells, medium, and bacterial suspension containing fluorophores to large numbers of 384 well dishes</td></tr><tr><td>Combi standard bore manifold</td><td>Thermo Fisher Scientific</td><td>24072670</td><td>Default predispense volume of 20 &amp;#956;l is insufficient to compensate for settling &amp;mdash; increase to 80 &amp;#956;l</td></tr><tr><td>White 384 well dishes treated for tissue culture</td><td>Corning</td><td>3570</td><td>For reading luminescence and fluorescence; Greiner catalog # 781080 also tested successfully</td></tr><tr><td>DMSO (tissue culture grade, in sealed ampoules)</td><td>Sigma-Aldrich</td><td>D2650</td><td>For dissolving positive control and test compounds</td></tr><tr><td>Azithromycin</td><td>Sigma-Aldrich</td><td>PHR1088</td><td>Antibiotic positive control</td></tr><tr><td>Saponin (from Quillaja bark)</td><td>Sigma-Aldrich</td><td>S-4521</td><td>Cytoxicity positive control</td></tr><tr><td>Multichannel pipettor</td><td>Thermo Fisher Scientific</td><td>Finnpipette</td><td>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><td>Epson pin transfer robot</td><td>Epson/ICCB-L</td><td>(Custom equipment)</td><td>For transfer of fixed amounts of test compounds from library arrays</td></tr><tr><td>D300 digital dispensing system</td><td>Hewlett-Packard via Tecan</td><td>D300</td><td>For transfer of variable amounts of test compounds ranging from 11 picoliters to 10 &amp;micro;l</td></tr><tr><td>T8+ cartridges for D300 digital dispensing system</td><td>Hewlett-Packard via Tecan</td><td>T8+</td><td>For dispensing test compounds</td></tr><tr><td>Epifluorescence microscope with computer-connected digital camera</td><td>Nikon</td><td>Ti</td><td>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><td>Glass-bottom tissue culture dishes</td><td>MatTek Corporation</td><td>P35G-1.5-20-C</td><td>For live cell imaging. Dishes such as the MatTek allow microscopic visualization at 600X or 1,000X magnification through use of an inverted epifluorescent or confocal microscope.&amp;nbsp; 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><td>Photoshop CS6</td><td>Adobe</td><td></td><td>Adobe photoshop or similar programs can be used to pseudocolor and merge light microscopic and fluorescent images.</td></tr><tr><td>Mathematica 10</td><td>Wolfam</td><td></td><td>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 ...

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

Research

JoVE Journal

Immunology and Infection

7.8K Views.  Beth Israel Deaconess Medical Center. 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.

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

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 ...

A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug screening assays utilize cytotoxicity as one of the critical characteristics to be profiled for individual compounds. Cells in culture are a useful model to assess cytotoxicity before proceeding to follow up on promising lead compounds in more costly and labor-intensive animal models. We describe a strategy to identify compounds that affect cell growth in a tdTomato expressing human neural stem cells (NSC) line. The strategy uses two complementary assays to assess cell number. One assay works via the reduction of 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as a proxy for cell number and the other directly counts the tdTomato expressing NSCs. The two assays can be performed simultaneously in a single experiment and are not labor intensive, rapid, and inexpensive. The strategy described in this demonstration tested 57 compounds in an exploratory primary screen for toxicity in a 96-well plate format. Three of the hits were characterized further in a six-point dose response using the same assay set-up as the primary screen. In addition to providing excellent corroboration for toxicity, comparison of results from the two assays may be effective in identifying compounds affecting other aspects of cell growth.

It is often necessary to assess the potential cytotoxicity of a set of compounds on cultured cells. Here, we describe a strategy to reliably screen for toxic compounds in a 96-well format.

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug ...

Biochemistry

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells&#39; mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes &#8211; Hoechst 33342 and propidium iodide.

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...

Cancer Research

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Medicine

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

Neuroscience

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 High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

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

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