an in vitro fluorescence-based assay to measure plasma membrane resealing efficiency

An In Vitro Fluorescence-Based Assay to Measure Plasma Membrane Resealing Efficiency

To set up a high throughput fluorescence-based assay for the repair permissive conditions, wash the cells two times with 200 microliters of 37 degrees Celsius M1 medium per well, and add 100 microliters of fresh 37 degrees 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 degrees Celsius M2 medium supplemented with 5-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 degrees 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 5 minutes prior to the start of the kinetic assay when plate 1 is on ice and plate 2 is being prepared.
When the first plate is finished imaging, immediately transfer the plate onto a sheet of aluminum foil on ice. After 5 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 1 minute, and immediately transfer the first plate to the plate reader for the postkinetic assay using the spectrofluorometer mode. At the end of the kinetic assay, immediately acquire a postkinetic imaging of the plate image as demonstrated for the prekinetic imaging.

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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 cells.
	<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 a 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 95 g of Hanks Balanced Salt Solution, 0.476 g of MgCl2 (5 mM), and 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&#39;,N&#39;, 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-minute 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, 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-minute 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 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 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>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>SpectraMax i3x Multi-Mode Microplate Reader</td>
			<td>Molecular Devices</td>
			<td>i3x</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMax 300 Imaging cytometer</td>
			<td>Molecular Devices</td>
			<td>5024062</td>
			<td></td>
		</tr>
		<tr>
			<td>TO-PRO-3</td>
			<td>ThermoFisher Scientific</td>
			<td>T3605</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>ThermoFisher Scientific</td>
			<td>P3566</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa</td>
			<td>ATCC</td>
			<td>CCL2</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa H2B-GFP</td>
			<td>Millipore</td>
			<td>SCC117</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25%</td>
			<td>ThermoFisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Corning flat bottom black polystyrene tissue culture treated plate</td>
			<td>Corning</td>
			<td>3603</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; balanced Salts</td>
			<td>Sigma-Aldrich</td>
			<td>H4891</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>ISC BioExpress</td>
			<td>0732-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose, HybriMax</td>
			<td>Sigma-Aldrich</td>
			<td>G5146-1KG</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Lam, J. G., et al. <a href="https://app.jove.com/v/58351">High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells.</a> J. Vis. Exp. (2019).
This video demonstrates an assay to study the plasma membrane resealing efficiency in mammalian cells. The cells are exposed to a bacterial pore-forming toxin, which forms pores in the cell membrane. In the presence of extracellular calcium ions, the pore causes calcium influx, inducing membrane-resealing events. The resealing efficiency is assessed using DNA-binding propidium iodide dye, which only enters cells without a resealed membrane.

<img alt="Figure 1" src="/files/ftp_upload/21981/21981_Figure1.jpg" /> 
Figure 1: Experimental design. The flow diagram depicts a representative plate design configured to test the effect of seven test conditions in comparison to control non-treated cells. Additional controls should be included if appropriate, as for example drug vehicles. Cells are plated (plate 1) 24 h prior to the experiment. On the day of the experiment, cells in plate 1 are washed with M1 or M2 ...

1. Preparation

	Cell Plating
	Note: Human cervical epithelial cells, HeLa and HeLa expressing Histone 2B-GFP (H2B-GFP), were used in this protocol, but ...

Take a microplate containing mammalian cells on ice.
Add a medium containing calcium to selected wells and a medium without calcium to the others.
Introduce monomers of a bacterial pore-forming toxin. A low temperature prevents monomer aggregation, facilitating binding to cell membrane cholesterol.
Incubate at a physiological temperature, allowing the cholesterol-bound monomers to oligomerize and insert into the membrane, forming a pore.
In wells with extracellular calcium, the pore causes calcium influx.
The influx triggers exosomal shedding of the toxin and membrane resealing.
The intracellular calcium increase also induces lysosomal exocytosis, releasing an enzyme that converts the membrane's sphingomyelin to ceramide.
The ceramide-enriched membrane triggers toxin endocytosis, resealing the membrane.
Membrane resealing is hindered in wells without extracellular calcium.
Add propidium iodide or PI &#8212; a fluorescent dye &#8212; that cannot enter cells with a repaired membrane but enters damaged cells to bind DNA, emitting fluorescence.
Measure fluorescence at defined intervals to assess the membrane resealing efficiency.

20 Aufrufe. Ein in vitro fluoreszenzbasierter Assay zur Messung der Effizienz der Wiederversiegelung von Plasmamembranen

Serie: Ein in vitro fluoreszenzbasierter Assay zur Messung der Effizienz der Wiederversiegelung von Plasmamembranen - Experiment

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

Echtzeit Live-Zelle Durchflusszytometrie, Calcium Zustrom, Porenbildung und Phagozytose durch P2X7-Rezeptoren im Erwachsenen neurale Vorläuferzellen zu untersuchen

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Forschung

JoVE Journal

Entwicklungsbiologie

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can result in disease. Understanding the underlying molecular mechanisms surrounding cell membrane repair is, therefore, an important objective to the development of novel therapeutic strategies for diseases associated with dysfunctional cell membrane dynamics. Many in vitro and in vivo studies aimed at understanding cell membrane resealing in various disease contexts utilize two-photon laser ablation as a standard for determining functional outcomes following experimental treatments. In this assay, cell membranes are subjected to wounding with a two-photon laser, which causes the cell membrane to rupture and fluorescent dye to infiltrate the cell. The intensity of fluorescence within the cell can then be monitored to quantify the cell's ability to reseal itself. There are several alternative methods for assessing cell membrane response to injury, as well as great variation in the two-photon laser wounding approach itself, therefore, a single, unified model of cell wounding would beneficially serve to decrease the variation between these methodologies. In this article, we outline a simple two-photon laser wounding protocol for assessing cell membrane repair in vitro in both healthy and dysferlinopathy patient fibroblast cells transfected with or without a full-length dysferlin plasmid.

Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

Zellmembran Reparatur Assays mit einem zwei-Photonen-Laser-Mikroskop

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can ...

Immunologie und Infektion

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While biophysical approaches help understand pore formation, they often rely on reductionist approaches lacking the full complement of membrane lipids and proteins. Cultured human cells provide an alternative system, but their complexity and redundancies in repair mechanisms make identifying specific mechanisms difficult. In contrast, the human protozoan pathogen responsible for cutaneous leishmaniasis, Leishmania major,&#160;offers an optimal balance between complexity and physiologic relevance. L. major is genetically tractable and can be cultured to high density in vitro, and any impact of perturbations on infection can be measured in established murine models. In addition, L. major synthesizes lipids distinct from their mammalian counterparts, which could alter membrane dynamics. These alterations in membrane dynamics can be probed with PFTs from the best-characterized toxin family, cholesterol-dependent cytolysins (CDCs). CDCs bind to ergosterol in the Leishmania membrane and can kill L. major promastigotes, indicating that L. major is a suitable model system for determining the cellular and molecular mechanisms of PFT function. This work describes methods for testing PFT function in L. major promastigotes, including parasite culture, genetic tools for assessing lipid susceptibility, membrane binding assays, and cell death assays. These assays will enable the rapid use of L. major as a powerful model system for understanding PFT function across a range of evolutionarily diverse organisms and commonalities in lipid organization.

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

Presented here is a protocol using Leishmania major promastigotes to determine the binding, cytotoxicity, and signaling induced by pore-forming toxins. A proof-of-concept with streptolysin O is provided. Other toxins can also be used to leverage the genetic mutants available in L. major to define new mechanisms of toxin resistance.

Entschlüsselung des molekularen Mechanismus und der Funktion von porenbildenden Toxinen mit Leishmania major

Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While ...

An In Vitro Fluorescence-Based Assay to Measure Plasma Membrane Resealing Efficiency

Transkript

An In Vitro Fluorescence-Based Assay to Measure Plasma Membrane Resealing Efficiency