Name: high-throughput measurement of plasma membrane resealing efficiency in mammalian cells
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells at JoVE.com

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 ...

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 ...

This assay was developed to address key questions in the field of plasma membrane biology and to establish how efficiently damaged cells can reseal their plasma membrane. This technique can be used to measure the efficiency of plasma membrane resealing in a high-throughput capacity and to identify specific proteins and corresponding pathways that mediate plasma membrane resealing. Begin by adding 20 milliliters of a 2.5 times 10 to the fifth HeLa cells per milliliter of growth medium suspension into a sterile pipette basin and use a 10 milliliter serological pipette to thoroughly mix the cells.Next, use a multichannel micropipette to seed 100 microliters of cells per well in triplicate in a 96-well flat, clear bottom, black polystyrene tissue culture treated plate. Remember that a homogenous cell distribution is key to a successful experiment as comparisons between all the experimental conditions require equivalent cell counts. After 24 hours in the humidified cell culture incubator at 37 degrees Celsius and 5%CO2, prewarm the plate reader to 37 degrees Celsius and set the optical configuration to monochromator, the read mode to fluorescence, and the read type to kinetic.Under the wavelength settings, select a nine nanometer excitation and a 15 nanometer emission bandpass. For assays using propidium iodide, set the excitation and emission wavelengths to 535 nanometers and 617 nanometers, respectively. Under plate type, select 96-wells for the plate format, and select a preset plate configuration corresponding to a black wall clear bottom plate.Under read area, highlight the wells that will be analyzed throughout the kinetic assay. Under PMT and optics, preset the flashes per read to six and check the box for read from bottom. Under timing, enter zero hours 30 minutes and zero seconds into the total run time for a 30 minute kinetic assay and enter zero hours five minutes and zero seconds for the interval.Confirm the specified settings in the settings information and click OK.Then click read to initiate the kinetic run. To set the imaging parameters within the settings mode, set MiniMax for the optical configuration, imaging for the read mode, and endpoint for the read type. Under wavelengths, select transmitted light and the appropriate fluorescence boxes corresponding to the experimental excitation and emission wavelengths.Set the plate type and the read area as just demonstrated and under the well area setting, set the number of sites within a well to be imaged. Under the image acquisition settings for GFP, set the device to image the entire well with an exposure time of 20 milliseconds per image. For transmitted light, acquire a single image of the center of each well with an eight millisecond exposure time.For propidium iodide, acquire a single image at the center of each well with a 20 millisecond exposure time. Then confirm the settings in the settings information and click OK and read to initiate the imaging. To setup a high-throughput fluorescence-based assay for the repair permissive conditions, wash the cells two times with 200 microliters of 37 degree Celsius M1 medium per well and add 100 microliters of fresh 37 degree Celsius M1 medium supplemented with 30 micromolar propidium iodide after discarding the second wash.For repair restrictive conditions, wash the cells one time with 200 microliters of 37 degree Celsius M2 medium supplemented with five millimolar EGTA per well followed by one wash with 200 microliters of M2 medium alone. After discarding the second wash, add 100 microliters of fresh 37 degree Celsius M2 medium supplemented with 30 micromolar propidium iodide per well. Then image the plate under transmitted light, GFP, and PI as just demonstrated.While the plate is being imaged, place a 96 well round bottom polypropylene microplate on ice and configure the plate as just demonstrated for the previous plate. For repair permissive conditions, add 100 microliters of ice cold M1 medium supplemented with 60 micromolar propidium iodide per well followed by the addition of 100 microliters of ice cold M1 with or without 4X listeriolysin O per well. For repair restrictive conditions, add 100 microliters of ice cold M2 medium supplemented with 60 micromolar propidium iodide per well followed by the addition of 100 microliters of ice cold M2 with or without 4X listeriolysin O per well.It is imperative to prepare listeriolysin O at the appropriate experimental concentration on ice approximately five minutes prior to the start of the kinetic assay when plate one is on ice and plate two is being prepared. When the first plate is finished imaging, immediately transfer the plate onto a sheet of aluminum foil on ice. After five minutes, transfer 100 microliters from each well of the second plate to the appropriate corresponding well in the first cooled plate, inserting the pipette tips below the meniscus of solution in each well before ejecting the volume without mixing for proper distribution of the toxin.Allow the toxin to bind to the host cells for one minute and immediately transfer the first plate to the plate reader for the post-kinetic assay using the spectrofluorometer mode. At the end of the kinetic assay, immediately acquire a post-kinetic imaging of the plate image as demonstrated for the pre-kinetic imaging. To determine the cell count based on the nuclear fluorescence, in the microplate cell enumeration software under settings, select re-analysis, and in the image analysis settings, select discrete object analysis using 541 as a wavelength for finding objects.Within the find objects option, use the draw on images finding method, select nuclei, and click apply, then click OK and read to initiate the cell counting algorithm. GFP expression does not interfere with propidium iodide intensity measurements in the presence or absence of listeriolysin O treatment. The expression of propidium iodide can bleed through GFP fluorescence as evidenced by an increase in GFP fluorescence intensity in HeLA H2B-GFP cells in the post-kinetic imaging assay compared to the pre-kinetic analysis.Importantly however, this crossover does not affect cell counting, as the segmentation process involved in the enumeration of the nuclei is unaffected by an increase in GFP fluorescence. In the absence of listeriolysin O, the plasma membrane integrity remains constant in the presence or absence of extracellular calcium, while listeriolysin O treatment in the presence of calcium supplemented medium results in a steady increase in propidium iodide fluorescence intensity. In the absence of extracellular calcium however, there is a significantly steeper increase in propidium iodide fluorescence, reflecting the absence of membrane resealing.Alternatively, a carbocyanine nucleic acid binding dye with fluorescence in the far red can be substituted for propidium iodide to minimize the spectral overlap with GFP. In addition, the larger excitation coefficient for the far red dye exhibits a higher signal to noise ratio and a better resolution between the repair permissive and repair restrictive conditions. While attempting this procedure, it is recommended to label all of the tubes a day ahead of experiment, as this will prepare you for all of the reagent preparation on the day of the experiment.Following this procedure, other methods like image cytometry can be performed to answer additional questions, such as does a pore forming toxin damage all cells uniformly or are some cells more susceptible than others. After its development, this technique has paved the way for researchers in the fields of infectious disease and plasma membrane repair to explore the effects of pore size on the repair efficiency of different cell types.

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