We thank Naomi Ziv, Sasha Levy and Shuang Li for their contributions to developing this protocol, David Gresham for shared equipment, and Marissa Knoll for help with video production. This work was supported by National Institutes of Health grant R35GM118170.

1. Preparation of Randomized Plates (Prior to Experiment Day)
<ol>
	<li>Plan the strains and conditions to be tested with the growth assay. At this point, randomly assign strains and conditions to any well. 
	NOTE: When considering plate setup, it is advisable to include more than one replicate per strain and growth condition on a single plate to account for well-related noise in measurements. See Discussion for more details.</li>
	<li>Computationally randomize the location of each strain and environmental condition for plate replicates that will be run on different days.</li>
	<li>Grow all cells that will be used in the experiment to saturation in yeast extract-peptone-dextrose (YEPD; 2% glucose) medium in a shaker at 30 &#176;C (or any other appropriate temperature).</li>
	<li>Create the randomized stock plates either manually or with a liquid-handling robot. Add 10 &#181;L of the designated saturated cells to each well of a sterile U-bottom tissue culture plate. If multiple strains will be tested in a single well, do not combine them at this point; this combining will be done just prior to cell dilutions on the day of the experiment to ensure all strains are at the correct concentrations when plated as microcolony founder cells.</li>
	<li>Add 10 &#181;L of 30% glycerol to each well of each plate. Pipet up and down so that the cells and the glycerol become well mixed.</li>
	<li>Seal each plate with a foil cover and freeze down immediately at -70 &#176;C until ready to use. 
	&#8203;NOTE: It is important to create all randomized plates on the same day, and freeze them, so that the pre-growth conditions of the cells in each plate will have been identical and will not generate technical variation in the growth-rate assay.</li>
</ol>
2. Pre-Growth of Yeast
NOTE: Typically, this starts prior to experiment day and is highly dependent on the experimental question. See Discussion for details.
<ol>
	<li>Remove a stock plate (10 &#181;L yeast, 10 &#181;L glycerol per well) from the -70 &#176;C freezer and add 180 &#181;L of the media to be used for the experiment. If the experiment will be conducted using nutrient-limiting media, do not pre-grow yeast to saturation in the nutrient-limiting media as sporulation of yeast can occur. Instead, pre-grow in non-limiting media.</li>
	<li>Grow yeast while shaking at 30 &#176;C. Consider whether to run the assay starting with cells in log phase or in stationary phase to determine if diluting the cells multiple times prior to the experiment will be necessary. If the yeast strains or conditions in the assay are expected to have significantly different growth rates, then a two-day pre-growth period will be necessary in order for all the different conditions to reach stationary phase.</li>
</ol>
3. Microscope Setup
<ol>
	<li>Microscope plate preparation
	<ol>
		<li>Ensure that the microscope incubator is on and heating the microscope chamber to the desired growth temperature for the experimental conditions. For standard experiments using Saccharomyces cerevisiae cells, the incubator should heat the microscope chamber to 30 &#176;C to ensure that the growth conditions for the cells will be correct during the growth rate assay.</li>
		<li>Sanitize the workbench, pipettes, and other tools with 70% ethanol. Retrieve a microscope plate and place it on the bench on top of a lint- and static-free wipe. 
		NOTE: Never touch the bottom of the microscope plate, even with gloves on, and always set the microscope plate down on top of a lint- and static-free wipe any time it touches any surface. This prevents smudges or scratches from impeding growth-rate measurements once the experiment is being imaged.</li>
		<li>Thaw 5 mL of 5x concanavalin A solution, dilute to 1x with water, and filter sterilize through a syringe fitted with a 0.2-&#181;m filter.</li>
		<li>Filter sterilize all other liquids that will be used in the assay with a 0.2 &#181;m filter, including experimental media, to remove any crystals or debris that may have materialized in the solutions. The presence of crystals would reduce the quality of the microscopy images.</li>
		<li>Pipet 200 &#181;L of concanavalin A solution into each well of the microscope plate.</li>
		<li>Centrifuge the plate for 2 min at 411 x gravity (g) with a lint- and static-free wipe under the plate, to ensure that the concanavalin A solution evenly covers the bottom of each well and that there are no air bubbles.</li>
		<li>Cover the plate with its lid and let it sit for 1-2 h. The precise time the plate sits is flexible, but it is important to be consistent between different runs of the experiment.</li>
		<li>Remove all of the concanavalin A solution from the plate either by suction or by forcefully discharging it out into the sink or a receptacle. Be careful not to touch the glass part of the plate. It is acceptable if some drops of concanavalin A solution remain in the wells.</li>
		<li>Wash the microscope plate wells by adding 400 &#181;L of sterile water. Remove the water as done with the concanavalin A in the previous step. Do not let the plate sit dry.</li>
		<li>Immediately add 185 &#181;L experimental growth media into the plate. 15 &#181;L of correctly diluted cells will be added to this plate.</li>
	</ol>
	</li>
	<li>Yeast Cell Dilution 
	NOTE: The steps below describe a dilution of yeast from a saturated culture (approximately 108 cells/mL) 400-fold to achieve a concentration of 250,000 cells/mL, 15 &#181;L of which will be diluted into 400 &#181;L in the glass-bottom plate, giving a final number of approximately 4000 cells per well in a 96-well plate. If using a 384-well plate the final number of cells per well should be approximately 700 and the dilutions should be adjusted accordingly. This ratio should be adjusted for cells collected in log phase, growing in richer or poorer pre-growth media, or from different strains. The final density of cells per well should be reduced when running the growth rate assay for time periods longer than 10 h.
	<ol>
		<li>Set up two 96-well culture plates for serial dilutions: label as plate 1 and 2, and add 90 &#181;L experimental growth media (i.e., the media that the yeast will grow in on the microscope) to each serial dilution plate. 
		NOTE: Regardless of what final dilution is used, at least two serial dilutions of cells are recommended, in each of which a small volume of yeast is pipetted into a bigger volume of experimental media and then a large volume of experimental media is mixed in vigorously with a pipet (as in steps 3.2.5 and 3.2.6 below).</li>
		<li>Retrieve the plate of cells from pre-growth and centrifuge the plate for 2 min at 411 x g. 
		NOTE: It is very important not to cross-contaminate different wells in the plate. The purpose of this centrifugation step before removing the foil covering from plates is to ensure that yeast-filled droplets from one well do not fly off the foil and end up in other wells. Be careful never to tilt or agitate the plates to avoid yeast coming into contact with the foil covering after centrifugation.</li>
		<li>Carefully peel back the foil and resuspend cells by vigorously pipetting cells with a pipet set to approximately one-half the total volume in the plate while moving the pipet around the well to mix. Check that all cells have been resuspended from the bottom of the wells.</li>
		<li>If multiple strains within individual wells will be used, strains should be mixed at this time at the ratio necessary for the experiment. If a reference strain will be used to generate growth-rate measurements, the ratio of reference to test strain should be 1:1.</li>
		<li>Pipet 10 &#181;L of yeast from growth media into dilution plate 1. Add 100 &#181;L of experimental growth media to each well to a final volume of 200 &#181;L per well. Pipet up and down vigorously.</li>
		<li>Pipet 10 &#181;L of yeast from plate 1 into plate 2. Add 100 &#181;L of experimental growth media to each well, and pipet up and down vigorously to mix. 
		NOTE: These dilution steps are critical to help separate clusters of yeast that are stuck together at the end of the pre-growth stage and ensure that approximately equal numbers of yeast cells end up in each well. Having consistent numbers of yeast in each well helps remove experimental noise and biases in growth-rate measurements (see Representative Results).</li>
	</ol>
	</li>
	<li>Sonication 
	&#8203;NOTE: Sonication is optional, and only needs to be performed for yeast strains that have a high propensity to adhere to one another (e.g., some wild strains). For lab strains, sonication is generally not necessary and may be skipped by proceeding to step 3.4.
	<ol>
		<li>Sanitize a 96-pin sonicator head with 70% ethanol by placing it in a 96-well plate filled with 70% ethanol and dry with a lint- and static-free wipe.</li>
		<li>Set a sonication program that is sufficiently strong to break apart flocculated yeast cells, but does not kill cells or cause elevated stress responses. Some testing may be required to identify the best sonication program for a given experiment. The sonication program used in this experiment is: amplitude = 10, process time = 10 s, pulse-on = 1 s, pulse-off = 1 s. This exact program is likely not applicable to all sonicators so testing is suggested prior to the experiment day.</li>
		<li>Mix the yeast in serial dilution plate 2 once more by pipetting up and down vigorously five times.</li>
		<li>Place dilution plate 2 on the platform and secure it with the sonicator pins in the cell suspension but not touching the bottom of the plate. Run the sonication program using appropriate ear protection.</li>
		<li>After the program runs, clean the sonicator head with 70% ethanol and then with water, and then immediately proceed to microscope plate preparation so that the cells do not flocculate again.</li>
	</ol>
	</li>
	<li>Prepare Plate for Microscope:
	<ol>
		<li>Pipet 15 &#181;L of yeast from serial dilution plate 2 into the microscope plate to a volume of 200 &#181;L. Add 200 &#181;L of experimental growth media to each well to a final volume of 400 &#181;L per well, and pipet up and down vigorously to mix.</li>
		<li>Cover the plate with a breathable membrane. It is important to seal the plate well with this membrane, for example using a rubber roller.</li>
		<li>To adhere the yeast cells to the concanavalin A on the glass surface, centrifuge the plate with a lint- and static-free wipe beneath it for 2 min at 411 x g.</li>
		<li>At the microscope, wipe the top and bottom of the plate with a lint- and static-free wipe, and blow compressed air onto the plate to get rid of debris.</li>
		<li>Place the plate onto the microscope, making sure it is level and that the A1 well is in the top left corner.</li>
	</ol>
	</li>
</ol>
4. Time-lapse Microscopy Growth-rate Measurements
NOTE: During time-lapse microscopy the following features are computer controlled: x, y, and z position, shutters, and fluorescence filters. A hardware-based auto-focus system is optimal to prevent focal plane drift during time-lapse imaging. Alternatively, a software-based auto-focusing loop can be used. To maintain humidity in the microscope chamber, it is advised to keep a beaker with purified water in the chamber throughout the duration of the experiment.
<ol>
	<li>Create a list of positions (x,y) to image, so that each microscope-plate well is fully imaged. Avoid overlapping images so that no cell is analyzed multiple times.</li>
	<li>Image in brightfield with diascopic illumination (DIA) at a magnification of 15x. Set exposure to ~5 ms.</li>
	<li>Zoom in on the image digitally so that cells are clearly visible. Use the focusing knobs to identify the ideal focus for the experiment in the four wells on the corner of the plate and in one well at the center of the plate. Focus in such a way as to get maximum contrast of the cells.</li>
	<li>Set the z position (or autofocus position) for the experiment to be an average of the z/autofocus positions identified for each of these wells. If the microscope plate is well made and the glass bottom does not have defects, the ideal focus positions should be similar for each well. 
	NOTE: When analyzing images with the Processing Images Easily (PIE) image analysis pipeline11, 12, it is helpful for the cells to be slightly out of focus on the microscope so that there is a dark rim outside of the cell and a light-colored interior, which aids in accurate colony recognition and size estimates.</li>
	<li>If using fluorescent strains, identify the channels and the exposures with which to image, ensuring that no pixels are overexposed. When setting exposure time for fluorescent channels, turn off the &#34;live capture&#34; mode on the microscope to avoid exposing cells to fluorescence excitation for long periods of time, as this can both photobleach the cells and cause stress.</li>
	<li>Set up the time-sequence acquisition to capture images at the desired time interval for the desired length of time.</li>
	<li>Run the experiment.</li>
</ol>

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The authors have nothing to disclose.

The protocol described here is a versatile assay that allows cell growth and gene expression to be monitored simultaneously at the level of individual microcolonies. Combining these two modalities yields unique biological insights. For example, previous work has used this assay to show a negative correlation between expression of the TSL1 gene and microcolony growth rate in isogenic wildtype cells by measuring both simultaneously7,10. It is also possible...

Growth phenotypes contribute critically to yeast fitness. Natural selection can efficiently distinguish between lineages with growth rates differing by the inverse of the effective population size, which can exceed 108 individuals1. Furthermore, variability of growth rates among individuals within a population is an evolutionarily relevant parameter, as it can serve as the basis for survival strategies such as bet hedging2,3,4,5,6. Therefore, assays that allow for highly accurate measurements of growth phenotypes and their distributions are pivotal for the study of microorganisms. The microcolony growth assay described here can generate individual growth-rate measurements for ~105 microcolonies per experiment. This assay therefore provides a powerful protocol to study yeast evolutionary genetics and genomics. It lends itself particularly well to testing how variability within populations of genetically identical single cells is generated, maintained, and contributes to population fitness7,8,9,10.
The method described here (Figure 1) uses periodically captured, low-magnification brightfield images of cells growing in liquid media on a 96- or 384-well glass-bottom plate to track growth into microcolonies. The cells adhere to the lectin concanavalin A, which coats the bottom of the microscope plate, and form two-dimensional colonies. Because the microcolonies grow in a monolayer, microcolony area is highly correlated with cell number7. Therefore, accurate estimates of microcolony growth rate and lag time can be generated with custom image-analysis software that tracks the rate of change of the area of each microcolony. Furthermore, the experimental setup can monitor the abundances and even the subcellular localizations of fluorescently labeled proteins expressed in these microcolonies. Downstream processing of data from this microcolony growth assay can be achieved by custom analysis or by existing image-analysis software, such as Processing Images Easily (PIE)11, an algorithm for robust colony area recognition and high-throughput growth analysis from low-magnification, brightfield images, which is available via GitHub12.
Because growth-rate estimates derived from the microcolony-growth assay are generated from a large number of single-colony measurements, they are extremely accurate, with standard errors several orders of magnitude smaller than the estimates themselves for a reasonably sized experiment. Therefore, the power of the assay to detect growth-rate differences between different genotypes, treatments, or environmental conditions is high. The multiwell-plate format allows numerous different environment and genotype combinations to be compared in a single experiment. If strains constitutively express different fluorescent markers, they may be mixed in the same well and distinguished by subsequent image analysis, which could increase power further by allowing well-by-well data normalization.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62038/62038fig1v5.jpg" /> 
Figure 1: Schematic representation of the protocol. This protocol follows two main steps, which are the preparation of the experimental plate and the preparation of the cells to image. Randomization of plates and growth of cells should be conducted before and leading up to the experiment day. Repeated mixing of cells at each step during dilution is imperative in the steps until plating, and therefore preparing the experimental plate first is recommended so that it is ready for plating immediately upon the completion of cell dilution. <a href="https://www.jove.com/files/ftp_upload/62038/62038fig1v5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>General Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL Bottletop Filter .22 &#181;m PES Sterilizing, Low Protein Binding, w/45mm Neck</td>
			<td>Fisher</td>
			<td>CLS431154</td>
			<td>used to filter the media</td>
		</tr>
		<tr>
			<td>BD Falcon*Tissue Culture Plates, microtest u-bottom</td>
			<td>Fisher</td>
			<td>08-772-54</td>
			<td>96-well culture tubes used to freeze cells, pre-grow cells, and dilutions</td>
		</tr>
		<tr>
			<td>BD Syringes without Needle, 50 mL</td>
			<td>Fisher</td>
			<td>13-689-8</td>
			<td>Used to filter the Concanavalin A</td>
		</tr>
		<tr>
			<td>Costar Sterile Disposable Reagent Reservoirs</td>
			<td>Fisher</td>
			<td>07-200-127</td>
			<td>reagent reservoirs used to pipette solutions with multichannel pipette</td>
		</tr>
		<tr>
			<td>Costar Thermowell Aluminum Sealing Tape</td>
			<td>Fisher</td>
			<td>07-200-684</td>
			<td>96-well plate seal for pre-growth and freezing</td>
		</tr>
		<tr>
			<td>lint and static free Kimwipes</td>
			<td>Fisher</td>
			<td>06-666A</td>
			<td>lint and static free wipes to keep microscope plate bottom free of debris and scratches</td>
		</tr>
		<tr>
			<td>Nalgene Syringe Filters</td>
			<td>ThermoFisher Scientific</td>
			<td>199-2020</td>
			<td>0.2 &#956;m pore size, 25 mm diameter; used to filter concanavalin A solution</td>
		</tr>
		<tr>
			<td>Media Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Minimal chemically defined media (MD; 2% glucose)</td>
			<td></td>
			<td></td>
			<td>alternative microscopy media used for yeast pre-growth and growth during microscopy</td>
		</tr>
		<tr>
			<td>Synthetic Complete Media (SC; 2% glucose)</td>
			<td></td>
			<td></td>
			<td>microscopy media used for yeast pre-growth and growth during microscopy</td>
		</tr>
		<tr>
			<td>Yeast extract-peptone-dextrose (YEPD; 2% glucose) medium</td>
			<td></td>
			<td></td>
			<td>cell growth prior to freezing down randomized plates</td>
		</tr>
		<tr>
			<td>Microscopy Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Breathe-Easy sealing membrane</td>
			<td>Millipore Sigma</td>
			<td>Z380059-1PAK</td>
			<td>breathable membranes used to seal plate during microscopy experiment. At this stage breathable membranes are reccomended because they prevent condensation in the wells and allow for better microscopy images</td>
		</tr>
		<tr>
			<td>Brooks 96-well flat clear glass bottom microscope plate</td>
			<td>Dot Scientific</td>
			<td>MGB096-1-2-LG-L</td>
			<td>microscope plate</td>
		</tr>
		<tr>
			<td>Concanavalin A from canavalia ensiformis (Jack Bean), lyophilized powder</td>
			<td>Millipore Sigma</td>
			<td>45-C2010-1G</td>
			<td>Make 5x concanavalin A solution and freeze 5ml of 5x concanavalin A in 50 mL conical tubes at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Strains Used</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MAH.5, MAH.96, MAH.52, MAH.66, MAH.11, MAH.58, MAH.135, MAH.15, MAH.44, MAH.132</td>
			<td></td>
			<td></td>
			<td>Haploid mutation accumulation strains in a laboratory background, described in Hall and Joseph 2010</td>
		</tr>
		<tr>
			<td>EP026.2A-2C</td>
			<td></td>
			<td></td>
			<td>Progeny of the ancestral Hall and Joseph 2010 mutation accumulation strain, transformed with YFR054c&#916;::Scw11P::GFP</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Misonix Sonicator S-4000 with 96-pin attachment</td>
			<td></td>
			<td></td>
			<td>Sonicator https://www.labx.com/item/misonix-inc-s-4000-sonicator/4771281</td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti-E with Perfect Focus System</td>
			<td></td>
			<td></td>
			<td>Inverted microscope with automated stage and autofocus system</td>
		</tr>
	</tbody>
</table>

high-throughput live imaging of microcolonies to measure heterogeneity in growth and gene expression

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 105&#160;Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains&#39; average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.

The novelty of this protocol is that growth rate can be calculated for individual cells within a population by tracking their growth into microcolonies through time-lapse imaging (Figure 2A). Because microcolonies grow for many hours in a planar manner due to the presence of concanavalin A, their areas can be tracked throughout the experiment, and a linear fit to the change in the natural log of the area over time can be used to calculate growth rate for each individual colony observed <sup ...

Watch this Scientific Journal Video about High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression at JoVE.com

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth.

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...

high-throughput-live-imaging-microcolonies-to-measure-heterogeneity

Research

JoVE Journal

Biology

4.7K Views.  New York University. Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth. 

Video: High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...