We acknowledge Dr. Jesse Kwiek (The Ohio State University) for kindly allowing us to use his multi-mode detection platform for some preliminary experiments. Research reported in this article was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number RO1AI107250 to Stephanie Seveau. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Preparation
<ol>
	<li>Cell Plating 
	Note: Human cervical epithelial cells, HeLa and HeLa expressing Histone 2B-GFP (H2B-GFP), were used in this protocol, but this assay can be adapted to other mammalian cells19.
	<ol>
		<li>Detach adherent cells from a 75 cm2 cell culture flask by washing the cells with 2 mL of Trypsin-EDTA 0.25%. Replace the used trypsin with 2 mL of fresh trypsin-EDTA 0.25%.</li>
		<li>Incubate the cells at 37 &#730;C for 5 min until the cells have rounded and detached from the flask.</li>
		<li>Resuspend the cells in 8 mL of growth medium (DMEM containing 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 &#181;g/mL streptomycin).</li>
		<li>Determine the cell concentration using a hemocytometer and 10 &#181;L of cell suspension.</li>
		<li>Dilute the cells in growth medium to a concentration of 2.5 x 105 cells/mL.</li>
		<li>Pour the cell suspension into a sterile pipette basin and thoroughly mix the suspension using a 10 mL serological pipette.</li>
		<li>Using a 12-multichannel micropipette and 200 &#181;L tips, distribute HeLa cells (2.5 x 104 cells/100 &#181;L/well) in triplicate (or quadruplicate) in a 96-well flat, clear bottom, black polystyrene tissue culture-treated plate. 
		Note: A plating arrangement is presented as an example in Figure 1.</li>
		<li>Culture the cells for 24 h in a humidified cell culture incubator at 37 &#730;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Stock Solution Preparation
	<ol>
		<li>Prepare 1 L of a 10x stock of buffer M (used to prepare M1 and M2) by adding&#160;95 g of Hanks Balanced Salt Solution,&#160;0.476 g of MgCl2 (5 mM), and&#160;23.83 g of HEPES (100 mM) to 900 mL of water. Adjust the pH to 7.4 and raise the volume to 1 L. Filter sterilize.</li>
		<li>Prepare 50 mL of a 50x (1.25 M) stock of glucose by adding 11.26 g of D-(+)-Glucose to a total of 50 mL of water. Filter sterilize the solution.</li>
		<li>Prepare 50 mL of a 100x (120 mM) stock of calcium by adding 0.666 g of CaCl2 to a total of 50 mL of water. Filter sterilize the solution.</li>
		<li>Prepare 50 mL of a 10x (50 mM) stock of ethylene glycol-bis(2-aminoethylether)-N,N,N&#8217;,N&#8217;,tetraacetic acid (EGTA) by adding 0.951 g of EGTA to 40 mL of water. Increase the pH to 8 using NaOH to dissolve the EGTA, then raise the volume to 50 mL. Filter sterilize the solution.</li>
		<li>For a single 96-well plate, prepare 50 mL of Medium 1 (M1, contains Ca2+), 50 mL of Medium 2 (M2, Ca2+-free), and 15 mL of Medium 2 supplemented with EGTA, accordingly:
		<ol>
			<li>For M1, add 5 mL of 10x Buffer M, 0.5 mL of 100x CaCl2, and 1 mL of 50x glucose to 43.5 mL of water.</li>
			<li>For M2, add 5 mL of 10x Buffer M and 1 mL of 50x glucose to 44 mL of water.</li>
			<li>For M2/EGTA, add 1.5 mL of 10x Buffer M and 1.5 mL of 10x EGTA to 12 mL of water. 
			Note: All solutions containing propidium iodide (PI) should be prepared directly prior to adding to the cells.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Plate Reader/Imaging Cytometer Settings 
	Note: Use a multi-mode plate reader equipped with two detection units: a spectrofluorometer and an imaging cytometer. Limit the fluorescence exposure to avoid photobleaching the fluorophores.
	<ol>
		<li>Pre-warm the plate reader to 37 &#176;C before performing the assay.</li>
		<li>Set up the parameters for the kinetic assay accordingly within the Settings mode:
		<ol>
			<li>Choose Monochromator, FL (fluorescence), and Kinetic for the optical configuration, read modes, and read type, respectively.</li>
			<li>Under Wavelength Settings, select a 9 and 15 nm excitation and emission bandpass, respectively. For assays using propidium iodide (PI), set the excitation and emission wavelengths to 535 and 617 nm, respectively.</li>
			<li>Under Plate Type, select 96 Wells for the plate format and a pre-set plate configuration corresponding to a black-wall clear bottom plate.</li>
			<li>Under Read Area, highlight the wells that will be analyzed throughout the kinetic.</li>
			<li>Under PMT and Optics, preset the flashes per read to 6 and check the box for Read from Bottom.</li>
			<li>Under Timing, insert 00:30:00 in the Total Run Time box for a 30 min kinetic assay, and insert 00:05:00 for the Interval. 
			Note: For each time point and one wavelength, the reading time of a full 96-well plate is 30 s.</li>
			<li>Confirm the specified settings in the Settings Information to the right and select OK. Press Read to initiate the kinetic run.</li>
		</ol>
		</li>
		<li>Set up the imaging parameters accordingly within the Settings mode:
		<ol>
			<li>Choose Minimax, Imaging, and Endpoint for the optical configuration, read modes, and read type, respectively.</li>
			<li>Under Wavelengths, select transmitted light, and either or both the fluorescence boxes corresponding to excitation and emission wavelengths of 456/541 nm (GFP) and 625/713 nm (PI).</li>
			<li>Use the same options for the Plate Type and Read Area as defined in steps 1.3.2.3 and 1.3.2.4.</li>
			<li>Under Well Area Setting, select the number of sites within a well to be imaged. 
			Note: 12 sites correspond to a full-well image.</li>
			<li>Under the Image Acquisition Settings, select the exposure times for transmitted light,&#160;541 (GFP), and 713 (PI). For GFP, image the entire well with an exposure time of 20 ms/image. For transmitted light (TL) and PI fluorescence, acquire a single image of the center of each well with exposure times of 8 and 20 ms, respectively.</li>
			<li>Confirm the specified settings in the Settings Information to the right and select OK. The acquisition time for imaging the entire surface of each well (12 images/well) of a 96-well plate and for one wavelength is ~15 min. Press Read to initiate imaging. 
			Note: The acquisition time of a single image/well of a 96-well plate requires ~2.5 min/plate for one wavelength. The parameters described above correspond to the specific equipment in our laboratory. Spectrofluorometric measurements: A xenon flash lamp displaying 1.0 nm increment excitation wavelengths (250-850 nm) with an adjustable 9 or 15 nm bandpass, a photomultiplier tube detector with a &#62; 6 log dynamic range and an adjustable 15 or 25 nm emission bandpass. Imaging cytometer: An illumination light source capable of white light, 460 nm and 625 nm excitation wavelengths with a 20 nm bandpass, emission filters centered at 541 nm (108 nm bandpass) and 713 nm (123 nm bandpass), respectively, and a 4X objective coupled to a 1.25 megapixel 12-bit charge-coupled device camera.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Assay
Note: At the time of the assay, cells must be 70-90% confluent. During the wash steps, the medium should be removed from and applied to the side-wall of the well (not directly above the cells). Maintain the temperature of LLO at &#60; 4 &#730;C to prevent its aggregation until step 3.1.5.
<ol>
	<li>Prepare a stock of 30 &#181;M PI in M1 and a stock of 30 &#181;M PI in M2 pre-warmed at 37 &#730;C.</li>
	<li>Gently wash the cells in plate 1 using a 12-multichannel micropipette and 200 &#181;L tips, as follows:
	<ol>
		<li>For repair-permissive conditions, remove the growth medium and wash the cells twice with 200 &#181;L/well M1 pre-warmed at 37 &#730;C. Replace the medium with 100 &#181;L/well of warm M1 containing 30 &#181;M PI.</li>
		<li>For repair restrictive-conditions, remove the growth medium and wash the cells once with 200 &#181;L/well warm M2 containing 5 mM EGTA to chelate Ca2+, followed by one wash with 200 &#181;L/well M2. Replace the medium with 100 &#181;L/well warm M2 containing 30 &#181;M PI.</li>
		<li>After the growth medium has been washed and replaced with medium containing propidium iodide, directly move to step 2.1.3.</li>
	</ol>
	</li>
	<li>Image plate 1 under transmitted light, GFP, and PI as detailed under 1.3.3 (pre-kinetic). This step takes 15-20 min.</li>
	<li>During the 15 min period in step 2.1.3, prepare plate 2 using a 12-multichannel micropipette and 200 &#181;L tips as follows:
	<ol>
		<li>Place a 96-well round bottom polypropylene microplate on ice. Configure the plate using an experimental design corresponding to plate 1 (Figure 1).</li>
		<li>For repair-permissive conditions, add 100 &#181;L/well of ice-cold M1 containing 60 &#181;M PI, followed by the addition of 100 &#181;L/well of ice-cold M1 containing 4x LLO or not for the control.</li>
		<li>For repair-restrictive conditions, add 100 &#181;L/well of ice-cold M2 containing 60 &#181;M PI, followed by the addition of 100 &#181;L/well of ice-cold M2 containing 4x LLO or not for the control.</li>
	</ol>
	</li>
	<li>After imaging plate 1 (step 2.1.3), immediately place it on ice, using aluminum foil to separate the plate from direct contact with ice. Allow plate 1 to cool down for 5 min.</li>
	<li>Using a 12-multichannel micropipette and 200 &#181;L tips, transfer 100 &#181;L from each well into plate 2 (step 2.1.4) to the corresponding wells in the plate 1. To properly distribute the toxin in the media of plate 1, insert the tips below the&#160;meniscus and gently eject the volume without introducing bubbles. 
	Note: Do not pipette up and down, as this may inadvertently detach the cells.</li>
	<li>Leave the plate for an additional 1 min to allow the toxin to bind to the&#160;cells and immediately transfer plate 1 to the plate reader for the kinetic assay using the spectrofluorometer mode (step 1.3.2).</li>
	<li>At the end of the kinetic assay, immediately image plate 1 (post-kinetic) using step 1.3.3.</li>
</ol>
3. Analysis: Cell Enumeration
<ol>
	<li>Determine the cell count based on the nuclear fluorescence using the microplate cell enumeration software.
	<ol>
		<li>Within Settings, select Re-analysis, and under the category section within the Image Analysis Settings select Discreet Object Analysis using 541 as the wavelength for finding objects.</li>
		<li>Within the Find Objects option, using the Draw on Images finding method, select Nuclei under the settings tab, and press Apply.</li>
		<li>Press OK and Read to initiate the cell counting algorithm.</li>
	</ol>
	</li>
	<li>Alternatively, if no such tool is available, use an image analysis software such as ImageJ to enumerate cells.
	<ol>
		<li>In ImageJ, open the image file as a stack.</li>
		<li>Convert the stack to 8-bit greyscale images by clicking Image in the menu bar, hover over Type, and select 8-bit.</li>
		<li>Subtract the background: Click Image in the menu bar, hover over Adjust, and select Brightness/Contrast. Adjust the minimum value to remove the background noise and select Apply.</li>
		<li>Threshold to create binary images: Click Image in the menu bar, hover over Adjust, and select Threshold. Select Dark background, adjust the minimum and maximum threshold values, and click Apply.</li>
		<li>In the case of overlapping nuclei, a Watershed tool can be used to segment nuclei. Click Process in the menu, hover over Binary and select Watershed. 
		Note: This will automatically separate connected nuclei.</li>
		<li>Analyze the masked images by applying user-specified criteria (size and circularity) to refine the identification of nuclei and exclude cell debris.
		<ol>
			<li>Click on Analyze in the menu and then Analyze particles. Set the desired size (pixel^2) and circularity (a value of 1 is a perfect circle) ranges that are sufficient to include individual cells/nuclei.</li>
			<li>In the Show dropdown box, select the option(s) desired, check Summarize, and click OK to obtain cell counts.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Analysis: Kinetic Curves
<ol>
	<li>Transfer the kinetic data from the plate reader software to an analytical data software.</li>
	<li>For each experimental condition, average the fluorescence intensities of the replicates at each timepoint, along with the corresponding standard deviation and standard error of the mean for each experimental condition.</li>
	<li>For each experimental condition, trace the corresponding kinetic curve: PI intensity (y-axis) versus time (x-axis).</li>
	<li>To calculate the resealing efficiency of a given treatment condition, calculate the area under the curve (AUC) of the +LLO in M1 (AUC(M1)) and +LLO in M2 (AUC(M2)). Use the approach suggested below to assess the efficiency (E) of resealing: 
	<img alt="Equation" src="/files/ftp_upload/58351/58351eq1v2.jpg" /></li>
	<li>Perform a comparison between control and test treatment by determining the efficiency ratio (REff) indicated below: 
	<img alt="Equation 2" src="/files/ftp_upload/58351/58351eq2.jpg" /> 
	REFF = 1, test treatment has no effect on repair 
	REFF &#60; 1, test treatment inhibits repair 
	REFF &#62; 1, test treatment improves repair</li>
	<li>Calculate the area under the curve using the following equation: 
	<img alt="Equation 3" src="/files/ftp_upload/58351/58351eq3.jpg" />, where k is the total number of follow-ups.</li>
</ol>

The authors have nothing to disclose.

This assay measures the efficiency of membrane resealing at the cell population level with high-throughput capacity. It can be used to screen for cellular components or drug libraries that could affect membrane repair. The described assay used a 96-well plate format, but it can be adapted to 384-well plates for higher throughput. An advantage of this assay is its ability to obtain fluorescence measurements of adherent living cells in real time without the need for excessive cell processing such as cell detachment, fixati...

Mammalian cells are subject to mechanical, osmotic, and biochemical stress, resulting in the loss of plasma membrane integrity. Without rapid and efficient resealing, damaged cells would quickly succumb to programmed or necrotic death. Since the 1960s, efforts to understand the plasma membrane resealing process have been motivated by the devastating consequences associated with its dysfunctions. Indeed, diseases such as Limb-Girdle Muscular Dystrophy, diabetes, and Chediak-Higashi Syndrome have been linked to deficient plasma membrane repair due to mutations in the gene encoding dysferlin, production of advanced glycation end products, and defects in the lysosomal trafficking regulator CHS1, respectively1,2,3,4,5,6. However, to date, our understanding of membrane resealing is still limited7. Initial studies have demonstrated that membrane resealing is initiated by the influx of extracellular Ca2+ through the damaged plasma membrane8,9,10. Since then, several non-mutually exclusive Ca2+-dependent mechanisms have been proposed to reseal cells. The patch hypothesis proposes that in proximity to the wound, intracellular vesicles fuse with each other and the damaged plasma membrane to act as a patch11,12,13,14. A second model proposes that calcium-dependent exocytosis of lysosomes at the wound site releases the lysosomal enzyme acid sphingomyelinase, which converts sphingomyelin to ceramide in the outer leaflet of the plasma membrane. This sudden change in lipid composition results in ceramide-driven endocytosis of the damaged region15,16,17. Lastly, the third proposed mechanism involves a role for the endosomal sorting complex required for transport (ESCRT) to promote the formation of outward-facing vesicles that bud off from the plasma membrane18. Only a limited set of proteins was identified in these models, and their machinery must be further elucidated.&#160;
Here we describe a high-throughput assay that measures the plasma membrane resealing efficiency in adherent mammalian cells subjected to damage mediated by recombinant listeriolysin O (LLO)19. LLO is a pore-forming toxin (PFT) secreted by the facultative intracellular pathogen Listeria monocytogenes20,21,22 and belongs to the MACPF/CDC (membrane attack complex, perforin, and cholesterol-dependent cytolysin) superfamily. MACPF are mammalian pore-forming proteins involved in immune defenses, whereas CDCs are bacterial toxins mainly produced by Gram-positive pathogens that damage host cells to promote their pathogenic lifestyles23. CDCs are synthesized as water-soluble monomers or dimers that bind to cholesterol present in the plasma membrane and oligomerize into a prepore complex of up to 50 subunits. The prepore complex then rearranges to insert &#946;-strands across the lipid bilayer, forming a &#946;-barrel pore that spans 30-50 nm in diameter24,25,26,27.&#160; These large pores permit fluxes of ions and small cellular components in and out of the cell; though, some studies have proposed that pores of smaller sizes are also formed28,29,30. Among the CDCs, LLO displays unique properties including irreversible pH- and temperature-dependent aggregation, which is conducive to high-throughput analyses31,32. LLO can be added to the cell culture medium at 4 &#730;C, a temperature permissive to its binding to cells, but not to the formation of the pore complex. Initiation of pore formation can then be synchronized by raising the temperature to 37 &#730;C, allowing for the efficient diffusion of toxin molecules in the plane of the membrane to form oligomers and for the conformational remodeling involved in pore generation. Therefore, following the switch in temperature, the kinetic of cell damage will depend on the amount of toxin bound to the plasma membrane. Importantly, soluble LLO (not bound to the plasma membrane) rapidly and irreversibly aggregates when the temperature reaches 37 &#730;C, which alleviates the need to wash away unbound toxin molecules and limits the extent of membrane damage over time. Lastly, because LLO binds to cholesterol and forms pores in cholesterol-rich membranes, this assay is amenable to a wide range of mammalian cells. It is important to keep in mind that LLO affects host cell signaling mainly via pore formation, with a few exceptions in which pore-independent cell signaling may occur33,34,35,36,37,38,39. Therefore, it cannot be excluded that LLO signaling activities may influence the process of membrane repair.&#160;
This assay directly assesses the extent of cell wounding by measuring the incorporation of a cell impermeant fluorochrome (e.g., propidium iodide) that passively enters wounded cells and becomes highly fluorescent once it associates with nucleic acids. Hence, the fluorochrome can be maintained in the cell culture medium throughout the experiment, allowing real-time analyses of cell wounding. The fluorescence intensity of the nucleic acid-binding dye will increase with the concentration of toxin and, for a given concentration of toxin, will increase over time until all pores are formed, and cells are fully repaired or until saturation is reached. The influx of extracellular Ca2+ through membrane pores is a sine qua non event for resealing. Therefore, the resealing efficiency can be indirectly evidenced by comparing cell wounding in culture medium containing Ca2+ (repair permissive condition) to wounding in a Ca2+-free medium (repair restrictive condition). Because the fluorescence intensity of the nucleic acid-binding dye is directly proportional to the cell concentration in each well, it is important to seed cells at the same concentration in all wells. It is also important to enumerate cells in each well before and after the assay to ensure that cell detachment does not occur, as floating, aggregated cells can obscure fluorescence readings which may complicate data interpretation. To enumerate cells, cells expressing nuclear-localized histone 2B-GFP (H2B-GFP) were used in this assay. Temperature-controlled, multi-mode, microplate readers combine rapid, high-throughput measurements (using a 96 or 384-well plate format) of fluorescence intensities with microscopy imaging of living cells at 37 &#176;C. The latter can be used to enumerate cell number and observe the eventual formation of distinct cell populations.
Ultimately, this assay provides users the ability to expand their knowledge of the complexity of membrane repair mechanisms by screening for host molecules or exogenously added compounds that may control membrane repair. The following protocol describes the experimental steps to measure the resealing efficiency of cells exposed to LLO and evaluate the effects of a given drug or cellular treatment on resealing efficiency.&#160;

<table border="0" cellpadding="0" cellspacing="0" width="610">
	<tbody>
		<tr height="42">
			<td align="left" height="42" style="height:42px;width:216px;">SpectraMax i3x Multi-Mode Microplate Reader</td>
			<td align="left" style="width:108px;">Molecular Devices</td>
			<td style="width:119px;">i3x</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;width:216px;">MiniMax 300 Imaging cytometer</td>
			<td align="left" style="width:108px;">Molecular Devices</td>
			<td style="width:119px;">5024062</td>
			<td></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;width:216px;">TO-PRO-3</td>
			<td align="left" style="width:108px;">ThermoFisher Scientific</td>
			<td style="width:119px;">T3605</td>
			<td></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;width:216px;">Propidium Iodide</td>
			<td align="left" style="width:108px;">ThermoFisher Scientific</td>
			<td style="width:119px;">P3566</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:216px;">HeLa</td>
			<td align="left" style="width:108px;">ATCC</td>
			<td style="width:119px;">CCL2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:216px;">HeLa H2B-GFP</td>
			<td align="left" style="width:108px;">Millipore</td>
			<td style="width:119px;">SCC117</td>
			<td></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;width:216px;">Trypsin-EDTA 0.25%</td>
			<td align="left" style="width:108px;">ThermoFisher Scientific</td>
			<td style="width:119px;">25200056</td>
			<td></td>
		</tr>
		<tr height="63">
			<td align="left" height="63" style="height:63px;width:216px;">96-well Corning flat bottom black polystyrene tissue culture treated plate</td>
			<td align="left" style="width:108px;">Corning</td>
			<td style="width:119px;">3603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:216px;">Hanks&#39; balanced Salts</td>
			<td align="left" style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">H4891</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:216px;">EGTA</td>
			<td align="left" style="width:108px;">ISC BioExpress</td>
			<td style="width:119px;">0732-100G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="height:42px;width:216px;">HEPES</td>
			<td align="left" style="width:108px;">Fisher Scientific</td>
			<td style="width:119px;">BP310-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;width:216px;">D-(+)-Glucose, HybriMax</td>
			<td align="left" style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">G5146-1KG</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Cell counting accuracy: HeLa cells are frequently used as a model mammalian cell line to explore membrane repair mechanisms. When assessing membrane repair at the cell population level, it is important to plate cells at the same concentration in all wells for proper data interpretation. It is also important to verify at the time of the assay that cell numbers are equivalent across wells. HeLa cells that constitutively express histone 2B fused to GFP (H2B-GFP) were introduced in this assay...

Watch this Scientific Journal Video about High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells at JoVE.com

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

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 ...

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Immunology and Infection

7.7K Views.  The Ohio State University. 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.

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

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

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, ...

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2. Manual FV assays have been described 3, 4, but they are time consuming and subjective. Automated FV assays have been reported 5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9. Microplate assays have been reported for clot lysis 10, platelet aggregation 11, and coagulation Factors 12, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma.
This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) 13. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only &mu;l quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity).
Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14. DIC is associated with a poor prognosis and increases mortality above the pre-existing pathology 15. The assay was used to show that in 9 patients with DIC, the FV 1-stage, 2-stage, and total activities were decreased, on average, by 54%, 44%, and 42%, respectively, compared with normal pooled human reference plasma (NHP).
The FV microplate assay is easily adaptable to measure the activity of any coagulation factor. This assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical settings. This information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for coagulation disorders, such as DIC.

This study describes a novel microplate assay that measures FV coagulation activity during fibrin clot formation in human plasma which has not been reported previously. The method uses a kinetic microplate reader to continuously measure the change in absorbance at 405nm during fibrin clot formation in human plasma.

 In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin ...

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 ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

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 ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

Reliable and High Efficiency Extraction of Kidney Immune Cells

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

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

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

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

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

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In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

High-throughput Detection Method for Influenza Virus

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

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

High-throughput Gene Tagging in Trypanosoma brucei

Reliable and High Efficiency Extraction of Kidney Immune Cells

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein