We thank Daniel May, Marc Chevrette, and Don Hoang for critical reading of the manuscript. This work, including the efforts of Cameron R. Currie, was supported by the University of Wisconsin-Madison, Office of the Vice Chancellor for Research and Graduate Education with funding from the Wisconsin Alumni Research Foundation, funding also provided by the National Institutes of Health Centers for Excellence for Translational Research (U19-AI109673-01). Reed M. Stubbendieck was supported by a National Library of Medicine training grant to the Computation and Informatics in Biology and Medicine Training Program (NLM 5T15LM007359). The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Informed consent was obtained from the donor&#39;s parents, and the Human Subjects Committee at the University of Wisconsin-Madison approved the study (Institutional Review Board [IRB] approval number H-2013-1044).
1. Sample Culture
NOTE: This procedure is used here for the study of interactions among bacteria isolates from the human nasal cavity. In principle, the following methods are applicable to any culture condition. Brain-heart-infusion broth (BHI) is used for general propagation of nasal bacteria. All plates are solidified using 1.5% agar. For this study, samples are taken from saline solution flushed into a donor&#39;s nose (nasal lavage), transferred into microcentrifuge tubes, and frozen at -80 &#176;C.
<ol>
	<li>Use standard culture techniques to plate 100 &#181;L of each of the thawed lavage samples onto BHI plates.</li>
	<li>Incubate the plates aerobically at 37 &#176;C for 1 week.</li>
	<li>After incubation, select &#8805;2 colonies of each distinct morphotype per plate and passage the isolates aerobically on BHI plates by streaking a colony from the initial plate onto a new BHI plate and incubating the plate at 37 &#176;C. Repeat until the bacterial cultures are pure. 
	NOTE: Bacterial isolates can be identified through colony morphology, gram-staining, 16S rRNA gene sequencing, or another method. However, knowing the isolate&#39;s identity is not necessary for continuing the protocol.</li>
	<li>Cryopreserve all bacterial isolates at -80 &#176;C after combining 1 mL of 50% glycerol with 1 mL of bacterial overnight culture (see section 3) in cryotubes.</li>
</ol>
2. 3D Printing Stamps
NOTE: Polycarbonate was selected as the stamping material due to its high glass transition temperature (147 &#176;C) that exceeds standard autoclave temperatures (121 &#176;C), which minimizes the potential for deformation after repeated uses.
<ol>
	<li>Load the 3D printer with polycarbonate filament.</li>
	<li>Apply white school glue (polyvinyl acetate) to the print bed to aid in the adhesion and minimize warping of the inoculation stamp during the print.</li>
	<li>Load the .STL model file (Supplementary Data File) for the inoculation stamp (Figure 1) into the 3D printer software.</li>
	<li>Print the inoculation stamp at a 290 &#176;C nozzle temperature, 60 &#176;C bed temperature, and layer height of 0.38 mm.</li>
	<li>Wrap the stamp in the aluminum foil and sterilize by autoclaving for 1.5 h on a gravity cycle with 15 min of drying. 
	NOTE: Though polycarbonate is hygroscopic, the stamps only retain approximately 0.5% water weight after autoclaving.</li>
</ol>
3. Preparation of Overnight Cultures
<ol>
	<li>Using a serological pipette, pipette 3 mL of sterile BHI broth into 14 mL culture tubes.</li>
	<li>Using a sterile 1 &#181;L inoculating loop, inoculate a bacterial colony into the broth. Swirl the loop to ensure the clump disperses into the broth. Vortex the culture tubes briefly before incubation.</li>
	<li>Incubate the culture tubes at 37 &#176;C overnight (~16 h) on a shaker at 250 rpm.</li>
	<li>Vortex to break up the clumps of cells once the bacterial cultures reach sufficient&#160;turbidity (OD600&#160;&#8805; 1).</li>
</ol>
4. Preparation of Bioassay Plates
NOTE: Bioassay plates are prepared in a laminar flow hood to maintain sterility.
<ol>
	<li>Prepare BHI media with 1.5% agar and sterilize by autoclaving according to manufacturer instructions.</li>
	<li>After autoclaving, cool the BHI media to 55 &#176;C in a temperature-controlled water bath.</li>
	<li>Using a serological pipette, pipette 3 mL of molten BHI media into each of the 12 wells on a 12 well plate. Ensure that the wells are as exact and even as possible. One liter of media will yield ~27 bioassay plates.</li>
	<li>Allow the agar to set overnight.</li>
</ol>
5. Inoculating Bioassay Plates with the Test Organism
NOTE: A test organism refers to the organism for which the production of inhibitory activity (e.g., antibiotic production) is determined using the co-culture interaction assay. For this experiment, the test organisms are Actinobacteria isolated from nasal lavages samples.
<ol>
	<li>Inoculate the test organism on a bioassay plate by inserting a sterile 10 &#181;L inoculating loop into the overnight culture and streaking a culture droplet over the left third of a plate well. 
	NOTE: It is recommended to only streak one-third of the well, as overgrowth of the well prohibits later inoculation of target organisms.</li>
	<li>Repeat until all 12 wells on the plate have been inoculated with the test organism.</li>
	<li>Incubate plates upside down at the appropriate temperature for 7 days. At higher temperatures (&#8805;37 &#176;C) or in drier climates, store the plates in a humid container to prevent the plates from drying out.</li>
</ol>
6. Preparation of Target Organisms
NOTE: A target organism refers to the organism whose inhibition status is determined using the co-culture interaction assay. For this experiment, the target organisms are Staphylococcus spp. isolated from nasal lavages samples.
<ol>
	<li>After incubating the bioassay plates for 6 days, prepare overnight cultures of the specified target organisms, as done above (see section 3).</li>
</ol>
7. Target Organism Inoculation
NOTE: After overnight incubation, ensure that the cultures are turbid (OD600&#160;&#8805; 1). Some bacterial cultures may flocculate at the bottom of the culture tube. Vortex the culture tubes to disperse clumps and assess the culture turbidity.
<ol>
	<li>Prepare the target plate by filling each well of an empty 12 well plate with 1.8 mL of BHI and 200 &#181;L of the target overnight culture.</li>
	<li>Unwrap and place a sterile inoculation stamp into the target plate. Gently swirl the cultures around in the wells, taking care to ensure that the cultures do not cross contaminate neighboring wells.</li>
	<li>Lift the inoculation stamp and ensure that there is a droplet of diluted target culture on each stamp tip.</li>
	<li>Prepare a monoculture control plate by placing the inoculation stamp on an uninoculated bioassay plate and gently rock the stamp so that a culture drop inoculates each well. Once the inoculation stamp is removed, a droplet of culture should be visible in the wells of the bioassay plate.
	<ol>
		<li>If any wells are not inoculated with the inoculation stamp due to uneven levels of media, spot 3 &#181;L of diluted overnight culture onto the right side of the wells using a pipette.</li>
	</ol>
	</li>
	<li>Inoculate the bioassay plates as done above (see step 7.4.1), but align the stamp so that the tips align with the right side of the 12 well plate. Ensure that the stamp does not contact the existing bacterial colony when inoculating the wells.</li>
	<li>Carefully remove the stamp and place back into the target plate.</li>
	<li>Repeat the inoculation for each bioassay plate until all the plates are inoculated.</li>
	<li>Incubate bioassay plates upside down at the appropriate temperature for 7 days.</li>
</ol>
8. Scoring
<ol>
	<li>After co-culturing the test and target organisms for 1 week, score the interactions based on the following visual assessment:
	<ol>
		<li>Score the wells with target organism growth that exhibits growth that is indistinguishable from the monoculture control as &#34;0&#34; (no inhibition) (Figure 2A,B).</li>
		<li>Score the wells with target organism that exhibits diminished growth compared to the control as &#34;1&#34; (weak inhibition) (Figure 2C).</li>
		<li>Score the wells where the target organism did not grow as &#34;2&#34; (strong inhibition) (Figure 2D).</li>
	</ol>
	</li>
</ol>

<ol>
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The authors declare no competing interests.

Antibiotics and other secondary metabolites that mediate microbial interactions are useful for a multitude of applications, including drug discovery. Herein, a protocol for co-culture assays to assess large numbers of microbial interactions is presented. These co-culture interaction assays are a simple, affordable, scalable, and high throughput means to investigate many pairwise combinations of microorganisms in tandem. Target organisms are spotted next to test organisms in a well of a 12 well plate using an inoculation ...

In nature, microorganisms rarely exist in isolation; consequently, they are constantly interacting with other organisms. Therefore, studying how microorganisms interact with each other is essential to understanding a multitude of microbial behaviors1. Microbial interactions can be mutualistic, commensal, or antagonistic. These in teractions can affect not only the microorganisms themselves but also the environments and hosts that the microorganisms colonize1,2.
Many scientists study microbial interactions to identify new antimicrobial molecules. One of the first clinically important antimicrobial molecules was found through the study of microbial interactions. Sir Alexander Fleming observed a contaminating Penicillium spp. isolate that inhibited the growth of a Staphylococcus strain, which led to the discovery of the commonly used antibiotic penicillin3. Characterization of the mechanisms that microorganisms use to antagonize their competitors remains a fruitful resource for the discovery of antimicrobial molecules. For example, it was recently shown that Streptomyces sp. strain Mg1 produces antibiotic linearmycins, which have a lytic and degradative activity against Bacillus subtilis4.
Further, a non-ribosomally synthesized peptide named lugdunin was recently discovered after the observation that nasal commensal Staphylococcus lugdunensis inhibits Staphylococcus aureus5. Studies have also shown that mutualistic interactions between microorganisms are equally as powerful as antagonistic interactions for the discovery of antimicrobial molecules. For example, many fungus-farming ants in the tribe Attini harbor symbiotic bacteria called Pseudonocardia on their exoskeleton that produces antifungal molecules to inhibit an obligate pathogen of their fungal crop6. As the study of microbial interactions has been beneficial for discovering antimicrobial molecules, the use of high throughput screens may result in the discovery of new antimicrobial molecules.
With respect to the cost and ease of performance, the methodologies used to study microbial interactions range from simple to complex. For instance, an agar plug assay is an inexpensive and simple method that can be used to investigate antagonism between multiple microorganisms7. However, an agar plug assay is not an efficient procedure and can be labor-intensive for many pairwise combinations.To assess the effects of microbially produced products on target isolates of interest in a high throughput manner, many laboratories use disk diffusion assays8. These assays are easy and inexpensive and can be scalable to higher numbers of samples7. However, this assay requires the generation of microbial extracts and may produce misleading results for certain combinations of target organisms and antibiotics, such as Salmonella and cephalosporins9.
The preceding approaches rely on isolated components to elicit a response in a target organism, instead of allowing microorganisms to interact with each other. This is of note because interactions between microbes may elicit the production of &#34;cryptic&#34; antimicrobial molecules that are not produced in monoculture. For instance, it was recently shown that the antimicrobial keyicin is only produced by a Micromonospora sp. when co-cultured with a Rhodococcus sp. that is isolated from the same sponge microbiome10. More complex interaction methodologies circumvent this potential monoculture hindrance. For instance, the iChip is useful for isolating rare and difficult to cultivate bacteria from environmental samples and allows for the observation of microbial interactions through growth in situ11. To investigate interactions in detail, matrix assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF-IMS) can be used. This approach provides detailed information on the composition and distribution of small molecules and peptides produced by interacting microbial colonies with high spatial resolution. MALDI-TOF-IMS&#160;has also been used in multiple studies of bacterial interactions to characterize the mechanisms of competition12,13,14,15. However, MALDI-TOF-IMS often requires laborious sample preparation, specialized expertise to operate the equipment, and expensive and specialized mass spectrometers. For these reasons, it is a difficult technique to use for high throughput studies. Thus, a simple, scalable, and high throughput co-culture assay for microbial interactions that overcomes many limitations of the above approaches would be beneficial.
Here, a protocol for high throughput microbial co-culture is presented. This assay is simple and easily incorporated into preexisting studies of microbial interactions. In contrast to many commonly used methods for the study of microbial interactions, our method is simple, inexpensive, and is amenable to investigating large numbers of interactions. These assays are not only easy to perform, but the materials are widely available from most laboratory suppliers or public resources (e.g., libraries and makerspaces). Consequently, this assay is advantageous as a first line of investigation to identify and parse interesting patterns among many pairwise combinations of microorganisms, which may be especially useful for the investigation of microbial ecology.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 &#956;L disposable polystyrene inoculating loops, blue</td>
			<td>VWR</td>
			<td>12000-806</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;L disposable polystyrene inoculating loops, yellow</td>
			<td>VWR</td>
			<td>12000-810</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well cell culture plate, sterile with lid</td>
			<td>Greiner bio-one</td>
			<td>665 180</td>
			<td></td>
		</tr>
		<tr>
			<td>14 mL polystyrene round bottom tube, 17 x 100mm style, nonpyrogenic, sterile</td>
			<td>Falcon</td>
			<td>352057</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 self standing screw cap tubes with caps, sterile</td>
			<td>USA scientific</td>
			<td>1420-9710</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL serological pipet</td>
			<td>Cell Treat</td>
			<td>229225B</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar, bacteriological</td>
			<td>VWR</td>
			<td>J637</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart Infusion Broth</td>
			<td>Dot Scientific</td>
			<td>DSB11000-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate filament, white, 3mm diameter</td>
			<td>Keene Village Plastics</td>
			<td>12.1-3MM-WH-581.2-1KG-R</td>
			<td></td>
		</tr>
		<tr>
			<td>School Glue</td>
			<td>Elmer&#39;s</td>
			<td>EPIE304</td>
			<td></td>
		</tr>
		<tr>
			<td>Taz 6 3D printer</td>
			<td>Lulzbot</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

high throughput co-culture assays for the investigation of microbial interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

Co-culture interaction assays can be used to understand microbial interactions, identify patterns of interest, and uncover microbial isolates with intriguing activities. In these assays, a test organism is monocultured on one side of a 12 well agar plate and incubated for 7 days. Subsequently, a target organism is spotted next to the test organism and the two microbes were co-cultured for 7 days before scoring for the growth phenotype of the target organism. The assays are scored based on...

Watch this Scientific Journal Video about High Throughput Co-culture Assays for the Investigation of Microbial Interactions at JoVE.com

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

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Immunology and Infection

9.5K Views.  University of Wisconsin-Madison. The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

Video: High Throughput Co-culture Assays for the Investigation of Microbial Interactions

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

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

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

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

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

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 ...

ScanLag: High-throughput Quantification of Colony Growth and Lag Time

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are affected both by the doubling time of the microorganism and by any delay in growth upon transfer from one condition to another, the lag. The ScanLag method enables the characterization of these two independent properties at the level of colonies originating each from a single cell, generating a two-dimensional distribution of the lag time and of the growth time. In ScanLag, measurement of the time it takes for colonies on conventional nutrient agar plates to be detected is automated on an array of commercial scanners controlled by an in house application. Petri dishes are placed on the scanners, and the application acquires images periodically. Automated analysis of colony growth is then done by an application that returns the appearance time and growth rate of each colony. Other parameters, such as the shape, texture and color of the colony, can be extracted for multidimensional mapping of sub-populations of cells. Finally, the method enables the retrieval of rare variants with specific growth phenotypes for further characterization. The technique could be applied in bacteriology for the identification of long lag that can cause persistence to antibiotics, as well as a general low cost technique for phenotypic screens.

ScanLag is a high-throughput method for measuring the delay in growth, namely lag time, as well as the growth rate of colonies for thousands of cells in parallel. Screening using ScanLag enables the discrimination between long lag-time and slow growth at the level of a single variant.

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

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 ...

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

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 ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.