We would like to thank the members of Orman Lab for their valuable inputs during this study. This study was funded by the NIH/NIAID K22AI125468 career transition award and a University of Houston startup grant.

1. Preparation of growth medium, ofloxacin solution and E. coli cell stocks
<ol>
	<li>Regular Luria-Bertani (LB) medium: Add 10 g/L of tryptone, 10 g/L of sodium chloride (NaCl) and 5 g/L of yeast extract in deionized (DI) water. Sterilize the medium by autoclaving.</li>
	<li>LB agar plates: Add 10 g/L of tryptone, 10 g/L of NaCl, 5 g/L of yeast extract and 15 g/L agar in DI water and sterilize the medium by autoclaving. At the desired temperature (~55 &#176;C), pour ~30 mL of agar medium into square plates (10 x 10 cm). Dry the plates and store at 4 &#176;C.</li>
	<li>Modified LB medium: Add 10 g/L of tryptone and 5 g/L of yeast extract in DI water. Sterilize the medium by autoclaving.&#160;</li>
	<li>Modified LB medium including an osmolyte: Mix 2x modified LB medium with a 2x osmolyte solution in equal volumes. To prepare 2x modified LB medium, add 20 g/L of tryptone and 10 g/L of yeast extract in DI water and sterilize by autoclaving. To prepare the 2x osmolyte solution, dissolve the osmolyte of interest (2x amount) in DI water, and then filter sterilize the solution. &#160; 
	NOTE: The osmolyte of interest and its concentrations can be determined from the Phenotype Microarray screening assays (see Protocol 4). However, the tested osmolyte and the associated 2x concentration can be adjusted depending on the nature of the research. For example, to study the impact of 1 mM sodium chloride, a 2x osmolyte solution would be a 2 mM sodium chloride solution.</li>
	<li>Ofloxacin stock solution (5 mg/mL): Add 5 mg of ofloxacin (OFX) salt in 1 mL of DI water. Add 10 &#181;L of 10 M sodium hydroxide to increase the solubility of OFX in water, and then filter sterilize the solution. Prepare aliquots and store at -20 &#176;C. 
	NOTE: Ofloxacin is a quinolone antibiotic that has been widely used for both growing and non-growing bacterial cells14,21. The minimum inhibitory concentration (MIC) of OFX for E. coli MG1655 cells is within the range of 0.039&#8211;0.078 &#956;g/mL19,20. Also note that other antibiotics such as ampicillin and kanamycin are commonly used in persister research. The choice of antibiotics is dependent on the nature of the study.</li>
	<li>E. coli MG1655 cell stocks: &#160;Inoculate 2 mL of regular LB medium with a single colony in a 14 mL test tube (snap-capped) and culture the cells in an orbital shaker at 250 rpm and 37 &#176;C. When the cells reach the stationary phase, mix 500 &#956;L of the cell culture with 500 &#956;L of 50% glycerol (sterile) in a cryogenic vial and store at -80 &#176;C.</li>
</ol>
2. Propagation of cells to eliminate pre-existing persisters
<ol>
	<li>To prepare an overnight culture, scrape a small amount of cells from a frozen cell stock with a sterile pipette tip (do not thaw the glycerol cell stock), and inoculate the cells in 2 mL of modified LB medium in a 14 mL snap-capped test tube. Culture the cells in an orbital shaker at 250 rpm and 37 &#176;C for 12 h.</li>
	<li>First propagation
	<ol>
		<li>After 12 h, transfer 250 &#181;L of overnight culture to 25 mL of fresh modified LB medium in a 250 mL baffled flask covered with a sterile aluminum foil.</li>
		<li>Grow the cell culture in an orbital shaker at 250 rpm and 37 &#176;C until cells reach the mid-exponential phase (OD600 = 0.5).</li>
		<li>Measure the optical density at 600 nm (OD600) using a microplate reader every 30 min.</li>
	</ol>
	</li>
	<li>Second propagation
	<ol>
		<li>At OD600 = 0.5, dilute 250 &#181;L of cell culture from the first flask into 25 mL of fresh modified LB medium in a 250 mL baffled flask.</li>
		<li>Grow the second cell culture in an orbital shaker at 250 rpm and 37 &#176;C until OD600=0.5.</li>
		<li>Measure the optical density every 30 min. 
		NOTE: An overnight culture may have a significant amount of persister cells4,5,22. The dilution/growth cycle method5 described above can be used to eliminate these pre-existing persisters before transferring the cells to microarrays. Their elimination can be validated by quantifying persister levels of overnight cultures with the assay described in Protocol 3. This method has been already validated in a previous study19.</li>
	</ol>
	</li>
</ol>
3. Validating the elimination of pre-existing persister cells
<ol>
	<li>After the second propagation (see step 2.3), dilute 250 &#181;L of the cell culture (OD600 = 0.5) in 25 mL of fresh modified LB medium in a 250 mL baffled flask. 
	NOTE: For controls, dilute 250 &#181;L of overnight culture (see step 2.1) in 25 mL of fresh modified LB medium in a 250 mL baffled flask. Before transferring the cells to the flask, the cell density of the overnight culture should be adjusted in fresh modified LB medium to obtain OD600 = 0.5.</li>
	<li>Add 25 &#181;L of OFX stock solution (5 mg/mL) into the cell suspension and shake the flask gently to make the assay culture homogeneous. The final concentration of OFX is 5 &#181;g/mL in the assay culture.</li>
	<li>Incubate the assay culture in an orbital shaker at 250 rpm and 37 &#176;C.</li>
	<li>At every hour during the treatment (including 0 h, the time point before adding OFX into the assay culture), transfer 1 mL of the assay culture from the flask to a 1.5 mL microcentrifuge tube.</li>
	<li>Centrifuge the assay culture in the microcentrifuge tube at 17,000 x g for 3 min.</li>
	<li>Carefully remove 950 &#181;L of the supernatant without disturbing the cell pellet.</li>
	<li>Add 950 &#181;L of phosphate-buffered saline (PBS) solution into the microcentrifuge tube.</li>
	<li>Repeat the washing steps 3.5-3.7 for 3x in total until the antibiotic concentration is below the minimum inhibitory concentration (MIC).</li>
	<li>After the final wash, resuspend the cell pellet in 100 &#181;L of PBS solution, resulting in a 10x concentrated sample.</li>
	<li>Take 10 &#181;L of the cell suspension and serially dilute six times in 90 &#181;L of PBS solution using a 96 well round bottom plate.</li>
	<li>Spot 10 &#181;L of diluted cell suspensions on antibiotic-free fresh agar plates. To increase the limit of detection, plate the remaining 90 &#181;L of cell suspension on a fresh agar plate.</li>
	<li>Incubate the agar plates at 37 &#176;C for 16 h, and then count the colony-forming units (CFUs). Account for the dilution rates while calculating the total number of CFUs in 1 mL of assay culture. Kill curves are generated by plotting the logarithmic CFU values with respect to the duration of antibiotic treatment. 
	NOTE: A 6 h OFX treatment is sufficient to obtain a biphasic kill curve for E. coli cells19,20. The persister level of propagated cells (as measured by CFU counts at 6 h) should be significantly less than that of the overnight culture. The procedures described in Protocol 2 and 3 can be performed with regular LB depending on the research design. &#160;</li>
</ol>
4. Microarray plate screenings 
<ol>
	<li>Preparing microarray-cell cultures
	<ol>
		<li>Transfer 250 &#181;L of exponential-phase cells to 25 mL of fresh modified LB medium in a 50 mL centrifuge tube. Gently mix the cell suspension to make it homogeneous. 
		NOTE: The exponential-phase cells (OD600 = 0.5) are obtained from the second propagation step (see step 2.3).</li>
		<li>Transfer the diluted cell suspension into a sterile 50 mL reservoir.</li>
		<li>Using a multichannel pipette, transfer 150 &#181;L of the cell suspension to each well of a microarray, i.e., a 96 well plate containing various osmolytes. &#160; 
		NOTE: Wells that do not have osmolytes serve as controls.</li>
		<li>Cover the microarray with a gas-permeable sealing membrane.</li>
		<li>Incubate the plate in an orbital shaker at 37 &#176;C and 250 rpm for 24 h. 
		NOTE: In these experiments, commercially available plates were used, such as Phenotype Microarrays (PM-9 and PM-10) that include a wide range of osmolytes, pH buffers and other chemicals in a dried state at various concentrations. These microarrays are in half-area 96 well plate formats. Culture volumes should be adjusted depending on the type of plates being used. Microarrays can also be generated manually (see below).</li>
	</ol>
	</li>
	<li>Manual preparation of microarray plates
	<ol>
		<li>Transfer 75 &#956;L of 2x osmolyte solutions into the wells of a half-area 96 well plate.</li>
		<li>Transfer 500 &#181;L of exponential-phase cells to 25 mL of 2x modified LB medium in a 50 mL centrifuge tube. Gently mix the cell suspension to make it homogeneous. 
		NOTE: The inoculation rate was adjusted to be consistent with the microarray screening protocol described in 4.1. &#160;</li>
		<li>Add 75 &#956;L of the cell suspension into each well of the half-area 96 well plate containing 2x osmolyte solutions.</li>
		<li>Incubate the plate in an orbital shaker at 37 &#176;C and 250 rpm for 24 h.</li>
	</ol>
	</li>
	<li>Preparing persister assay plates
	<ol>
		<li>Prepare 25 mL of modified LB medium containing 5 &#181;g/mL of OFX in a 50 mL centrifuge tube and transfer this medium to a sterile reservoir.</li>
		<li>Transfer 190 &#181;L of modified LB medium with OFX from the reservoir into each well of a generic flat-bottom 96 well plate (persister-assay plate) using a multichannel pipette.</li>
		<li>Remove the microarray from the shaker (after culturing for 24 h) and transfer 10 &#181;L of cell cultures from the microarray to the wells of the persister-assay plate, containing modified LB medium with OFX.</li>
		<li>Take 10 &#181;L of cell suspensions from the persister-assay plate and serially dilute three times in 290 &#181;L of PBS solution using a round-bottom 96 well plate and a multichannel pipette.</li>
		<li>Following the serial dilution, spot 10 &#181;L of all serially diluted cell suspensions on antibiotic-free fresh agar plates using a multichannel pipette. &#160;</li>
		<li>Incubate the persister-assay plate (prepared in step 4.3.3) in an orbital shaker at 37 &#176;C and 250 rpm for 6 h after covering the plate with a gas-permeable sealing membrane.</li>
		<li>After 6 h incubation in a shaker, take the persister-assay plate out and repeat the steps 4.3.4-4.3.5.</li>
		<li>Incubate the agar plates for 16 h at 37 &#176;C, and then count CFUs. The CFU levels before and 6 h after the antibiotic treatment enable to calculate the persister fraction in each well. The CFU counts before the OFX treatment also help assess the effects of osmolytes and on E. coli viability.</li>
	</ol>
	</li>
</ol>
5. Validating the identified conditions
<ol>
	<li>Transfer 250 &#181;L of the exponential phase cells from Step 2.3 to 25 mL of fresh modified LB medium containing the osmolyte identified from the microarray screening (see Protocol 4).</li>
	<li>Incubate the flask in an orbital shaker at 250 rpm and 37 &#176;C for 24 h.</li>
	<li>After 24 h, remove the flask from the shaker and transfer 250 &#181;L of the cell culture to 25 mL of fresh modified LB medium in a 250 mL baffled flask.</li>
	<li>Add 25 &#181;L of OFX stock solution (5 mg/mL) in the cell suspension and shake the flask gently to make the assay culture homogenous. Incubate the flask in a shaker at 37 &#176;C and 250 rpm.</li>
	<li>At every hour during the treatment, transfer 1 mL of the assay culture from the flask to a 1.5 mL microcentrifuge tube.</li>
	<li>Centrifuge the assay culture in the microcentrifuge tube at 17,000 x g for 3 min.</li>
	<li>Remove 950 &#181;L of supernatant and add 950 &#181;L of PBS.</li>
	<li>Repeat the washing steps 5.6 and 5.7 for 3x.</li>
	<li>After the final wash, resuspend the cell pellet in 100 &#181;L of PBS solution.</li>
	<li>Take 10 &#181;L of the cell suspension and serially dilute 6x in 90 &#181;L of PBS solution using a 96 well round bottom plate.</li>
	<li>Spot 10 &#181;L of the diluted cell suspensions on an antibiotic-free fresh agar plate. To increase the limit of detection, plate the remaining 90 &#181;L of cell suspension on a fresh agar plate.</li>
	<li>Incubate the agar plate at 37 &#176;C for 16 h, and then count CFUs.</li>
</ol>

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The authors have nothing to disclose.

The high throughput persister assay described here was developed to elucidate the effects of various chemicals on E. coli persistence. In addition to commercial PM plates, microarrays can be constructed manually as described in step 4.2. Moreover, the protocol presented here is flexible and can be used to screen other microarrays, such as drug panels and cell libraries, that are in 96 well plate formats. The experimental conditions including the growth phase, inoculation rate and medium can be adjusted to test t...

Bacterial cultures contain a small subpopulation of persister cells that are temporarily tolerant to unusually high levels of antibiotics. Persister cells are genetically identical to their antibiotic-sensitive kins, and their survival has been attributed to transient growth inhibition1. Persister cells were first discovered by Gladys Hobby2 but the term was first used by Joseph Bigger when he identified them in penicillin-treated Staphylococcus pyogenes cultures3. A seminal study published by Balaban et al.4 discovered two persister types: type I variants that are primarily formed by passage through the stationary phase, and type II variants that are continuously generated during the exponential growth. Persisters are detected by clonogenic survival assays, in which culture samples are taken at various intervals during antibiotic treatments, washed, and plated on a typical growth medium to count the surviving cells that can colonize in the absence of antibiotics. The existence of persisters in a cell culture is assessed by a biphasic kill curve4,5 &#160;where the initial exponential decay indicates the death of antibiotic-sensitive cells. However, the killing trend decreases over time, eventually leading to a plateau region which represents the surviving persister cells. &#160;
Persister cells have been associated with various diseases such as tuberculosis6, cystic fibrosis7, candidiasis8 and urinary tract infections9. Almost all microorganisms tested so far were found to generate persister phenotypes, including highly pathogenic Mycobacterium tuberculosis6, Staphylococcus aureus10, Pseudomonas aeruginosa7 and Candida albicans8. Recent studies also provide evidence of the rise of multidrug-resistant mutants from persister subpopulations11,12. Substantial efforts in this field have revealed that persistence mechanisms are highly complex and diverse; both stochastic and deterministic factors associated with the SOS response13,14, reactive oxygen species (ROS)15, toxin/antitoxin (TA) systems16, autophagy or self-digestion17 and ppGpp-related stringent response18 are known to facilitate persister formation.
Despite significant progress in understanding the persistence phenotype, the effects of osmolytes on bacterial persistence have not been fully understood. Since the maintenance of optimal osmotic pressure is a necessity for cells&#8217; growth, proper functioning and survival, an in-depth study of osmolytes could lead to potential targets for anti-persister strategies. Although laborious, high-throughput screening is a very effective approach for identifying metabolites and other chemicals that play a crucial role in the persistence phenotype19,20. In this work, we will discuss our published method19, where we have used microarrays, i.e., 96 well plates containing various osmolytes (e.g., sodium chloride, urea, sodium nitrite, sodium nitrate, potassium chloride), to identify osmolytes that significantly influence E. coli persistence.

<table border="0" cellpadding="0" cellspacing="0" style="width:1204px;" width="1202">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:334px;">14-ml test tube</td>
			<td style="width:201px;">Fisher Scientific</td>
			<td style="width:161px;">14-959-1B</td>
			<td style="width:507px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E. coli strain MG1655</td>
			<td>Princeton University</td>
			<td></td>
			<td>Obtained from Brynildsen lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flat-bottom 96-well plate</td>
			<td>USA Scientific</td>
			<td>5665-5161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gas permeable sealing membrane</td>
			<td>VWR</td>
			<td>102097-058</td>
			<td>Sterilized by gamma irradiation and free of cytotoxins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Half-area flat-bottom 96-well plate</td>
			<td>VWR</td>
			<td>82050-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LB agar</td>
			<td>Fisher Scientific</td>
			<td>BP1425-2</td>
			<td>Molecular genetics grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ofloxacin salt</td>
			<td>VWR</td>
			<td>103466-232</td>
			<td>HPLC &#8805;97.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenotype microarray (PM-9 and PM-10)</td>
			<td>Biolog</td>
			<td>N/A</td>
			<td>PM-9 and PM-10 plates contained various osmolytes and buffers respectively</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Round-bottom 96-well plate</td>
			<td>USA Scientific</td>
			<td>5665-0161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td>Certified ACS grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium nitrate</td>
			<td>Fisher Scientific</td>
			<td>AC424345000</td>
			<td>ACS reagent grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium nitrite</td>
			<td>Fisher Scientific</td>
			<td>AAA186680B</td>
			<td>98% purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Square petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875711A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone</td>
			<td>Fisher Scientific</td>
			<td>BP1421-500</td>
			<td>Molecular genetics grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Varioskan lux multi mode microplate reader</td>
			<td>Thermo Fisher Scientific</td>
			<td>VLBL00D0</td>
			<td>Used for optical density measurement at 600 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract</td>
			<td>Fisher Scientific</td>
			<td>BP1422-100</td>
			<td>Molecular genetics grade</td>
		</tr>
	</tbody>
</table>

high-throughput screening of chemical compounds to elucidate their effects on bacterial persistence

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. They are an important health concern as they have been associated with recurrent chronic infections. Although stochastic and deterministic dynamics of stress-related mechanisms are known to play a significant role in persistence, mechanisms underlying the phenotypic switch to/from the persistence state are not completely understood. While persistence factors triggered by environmental signals (e.g., depletion of carbon, nitrogen and oxygen sources) have been extensively studied, the impacts of osmolytes on persistence are yet to be determined. Using microarrays (i.e., 96 well plates containing various chemicals), we have designed an approach to elucidate the effects of various osmolytes on Escherichia coli persistence in a high throughput manner. This approach is transformative as it can be readily adapted for other screening arrays, such as drug panels and gene knockout libraries.

Figure 1 describes our experimental protocol. The dilution/growth cycle experiments (see Protocol 2) were adapted from a study conducted by Keren et al.5 to eliminate the persisters originating from the overnight cultures. Figure 2A is a representative image of agar plates used to determine CFU levels of cell cultures before and after OFX treatment. In these experiments, cells were cultured in modified LB medium with osmolytes in half-are...

Watch this Scientific Journal Video about High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence at JoVE.com

High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

In this method paper, we present a high-throughput screening strategy to identify chemical compounds, such as osmolytes, that have a significant impact on bacterial persistence.

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. ...

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Research

JoVE Journal

Immunology and Infection

3.9K Views.  University of Houston. In this method paper, we present a high-throughput screening strategy to identify chemical compounds, such as osmolytes, that have a significant impact on bacterial persistence.

Video: High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

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 Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

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

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

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

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

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

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

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

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

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

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

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Automated, High-Throughput Detection of Bacterial Adherence to Host Cells

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in bacterial infections and constitutes a hallmark of potential threat. Therefore, examining the adherence of bacteria to host cells can be used as a component of bacterial threat assessment. A standard method for enumerating bacterial adherence to host cells is to co-incubate bacteria with host cells, harvest the adherent bacteria, plate the harvested cells on solid media, and then count the resultant colony forming units (CFU). Alternatively, bacterial adherence to host cells can be evaluated using immunofluorescence microscopy-based approaches. However, conventional strategies for implementing these approaches are time-consuming and inefficient. Here, a recently developed automated fluorescence microscopy-based imaging method is described. When combined with high-throughput image processing and statistical analysis, the method enables rapid quantification of bacteria that adhere to host cells. Two bacterial species, Gram-negative Pseudomonas aeruginosa and Gram-positive Listeria monocytogenes and corresponding negative controls, were tested to demonstrate the protocol. The results show that this approach rapidly and accurately enumerates adherent bacteria and significantly reduces experimental workloads and timelines.

Detection of host-bacterial pathogen interactions based on phenotypic adherence using high-throughput fluorescence labeling imaging along with automated statistical analysis methods enables rapid evaluation of potential bacterial interactions with host cells.

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in ...