Name: Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair (Video) | JoVE Journal
Uploaded: 2014-03-24T20:45:00.000+00:00
Duration: 16 min 44 s
Description: Learn to image cell membrane injury and repair using fluorescent dyes and live microscopy. This protocol reveals lysosomal exocytosis dynamics and subcellular mechanisms that enable injured cells to restore membrane integrity.

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5 minutes

This work was supported by National Institutes of Health Grants AR055686 and AR060836 and postdoctoral fellowship to AD by French Muscular Dystrophy Association (AFM). The cellular imaging facility utilized in this study is supported by National Institutes of Health Grants HD040677.

1. Imaging Cell Membrane Repair Using Bulk (Glass Bead) Wounding
This protocol allows separately marking the injured cells and those that fail to heal. Quantifying these populations of cells requires use of three conditions: 1. Test (C1) - Cells are allowed to repair in presence of Ca2+, 2. Control 1 (no injury C2) - Cells are incubated in presence of Ca2+, but not injured, and 3. Control 2 (no repair C3) - cells are allowed to repair in absence of Ca2+.
<ol>
	<li>Grow cells to &#62;50% confluence on three sterile coverslips and wash C1 and C2 twice with CIM at 37 &#176;C and C3 with PBS at 37 &#176;C and transfer coverslips on silicone O-rings.</li>
	<li>Add 100 &#956;l of prewarmed FITC dextran solution in CIM on C1 and C2 or in PBS on C3.</li>
	<li>Injure plasma membrane on C1 and C3 by gently rolling 40 mg glass beads over the coverslip at room temperature by manually tilting coverslips back and forth 6-8 times at an angle of 30&#176;. 
	Note: For mild injury, ensure the glass beads are spread uniformly, thus minimizing repeat injuries. To improve reproducibility of injury between samples, simultaneously carry out the glass bead injury of the different samples to be compared.</li>
	<li>Avoiding further rolling of beads, transfer all coverslips to a 37 &#176;C incubator at ambient CO2 and allow repair to proceed for 5 min.</li>
	<li>Without letting the beads roll, remove the glass beads and FITC dextran by washing with CIM (C1 and C2) or PBS (C3) at 37 &#176;C.</li>
	<li>Place coverslips back on the O ring and add 100 &#956;l of prewarmed lysine fixable TRITC dextran solution in CIM on C1 and C2 or in PBS on C3.</li>
	<li>Incubate at 37 &#176;C for 5 min at ambient CO2.</li>
	<li>Wash coverslips twice with prewarmed CIM and fix with 4% PFA for 10 min at RT.</li>
	<li>Wash twice with PBS and incubate for 2 min at RT in Hoechst dye.</li>
	<li>Wash samples twice with PBS, mount on a slide using mounting media and image cells using an epifluorescence microscope.</li>
	<li>Use cells from C2 to determine the background red and green staining intensity and use this value to threshold the red and green channels for all samples (C1-C3).</li>
	<li>Score total number of cells that are a) green (injured and repaired) and b) red or both red and green (injured, but failed to repair) in C1 and C3 samples.</li>
	<li>Count &#62;100 greens cells for each condition and express the fraction of cells that failed to repair as a percent of all cells injured (green and red).</li>
</ol>
2. Live Imaging of the Kinetics of Cell Membrane Repair Following Laser Injury
<ol>
	<li>Wash cells with prewarmed CIM and then put the coverslip in CIM with FM dye.</li>
	<li>Place the coverslip in a holder in the stage top incubator maintained at 37 &#176;C.</li>
	<li>Select a 1-2 mm2 region of the cell membrane, and irradiate for &#60;10 msec with the pulsed laser. Attenuate the laser power through the software to 40-50% of the peak power. Optimal power allows consistent, but nonlethal injury and this must be determined by trial and error for each individual instrument and cell line being used.</li>
	<li>To monitor repair, image every 10 sec in epifluorescence and brightfield, starting prior to injury and continue for 3-5 min&#160; following injury. 
	Note: For no repair control, repeat steps 2.1-2.4 with the cells injured in PBS containing FM dye.</li>
	<li>To quantify the kinetics of repair, measure cellular FM dye fluorescence and plot the change in intensity (&#916;F/F0) during the course of imaging. This data should be averaged for over 10 cells in each condition and plotted as averaged or individual cell&#39;s value, as needed.</li>
</ol>
3. Imaging Bulk (Glass Bead) Injury Induced Lysosomal Exocytosis
The samples include following cells grown to&#62;50% confluence: 1. Test (C1) - Cells allowed to repair in presence of Ca2+, 2. Control 1 (C2; No injury) - Cells neither injured nor incubated with primary antibody, and 3. Control 2 (C3; No repair) - Cells allowed to repair in absence of Ca2+.
<ol>
	<li>Wash coverslips C1 and C2 twice with CIM at 37 &#176;C and C3 with PBS at 37 &#176;C and transfer them on silicone O-rings.</li>
	<li>Add 100 &#956;l of prewarmed lysine fixable TRITC dextran in CIM on C1 and C2 and in PBS on C3.</li>
	<li>Injure plasma membrane on C1 and C3 as in step 1.3.</li>
	<li>Avoiding further rolling of beads, transfer all coverslips to a 37 &#176;C incubator at ambient CO2 and allow repair to proceed for 5 min.</li>
	<li>Remove the glass beads and TRITC dextran by washing the coverslips in cold growth medium, again ensuring no rolling of the glass beads on the cells.</li>
	<li>Rinse the coverslips twice with cold growth medium and transfer to the O-rings.</li>
	<li>To C1 and C3, add 100 &#956;l of rat anti mouse LAMP1 antibody in cold complete growth media and add the cold, complete growth media to C2.</li>
	<li>To allow antibody binding incubate coverslips for 30 min at 4 &#176;C.</li>
	<li>Wash the coverslips three times with cold CIM and fix all coverslips with 4% PFA for 10 min&#160; at RT and then rinse 3x with CIM.</li>
	<li>Incubate all coverslips in 100 &#956;l of blocking solution for 15 min at RT</li>
	<li>Incubate all coverslips in 100 &#956;l Alexa Fluor 488 anti rat antibody for 15 min at 4 &#176;C.</li>
	<li>Wash twice with PBS and incubate for 2 min at RT in Hoechst.</li>
	<li>Wash cells twice with PBS, mount on a slide using mounting media and image using an epifluorescence microscope.</li>
	<li>Using images of C2 cells, determine the nonspecific background staining in the red (TRITC dextran) and green (Alexa Fluor 488 antibody) channels and use these background staining values to threshold the red and green channels for all samples (C1-C3).</li>
	<li>Use the &#62;100 red labeled cells from C1 and C3 to measure the intensity of LAMP1 staining (green) in injured cells. For a successful experiment the LAMP1 staining in cells from C3 will be significantly lower than in cells from C1.</li>
</ol>
4. Live Imaging of Cell Membrane Injury Triggered Subcellular Trafficking
<ol>
	<li>Fluorescently label the compartment of interest by transfecting appropriate reporter (e.g.&#160;CD63-GFP for lysosomes8) or by using fluorescent dyes9.</li>
	<li>For labeling lysosomes with fluorescent dye, incubate cells grown to 50% confluence in growth media containing FITC-dextran</li>
	<li>Allow dextran to be endocytosed for 2 hr (or longer as needed for sufficient endosomal labeling with dextran by the cell line of interest) in the CO2 incubator.</li>
	<li>Wash cells with prewarmed growth media and incubate in it for 2 hr in a CO2 incubator to allow all endocytosed dextran to accumulate in the lysosome.</li>
	<li>Before imaging, rinse the coverslip in prewarmed CIM and mount in a coverslip holder on the stage top incubator at 37 &#176;C.</li>
	<li>Carry out widefield (for movement throughout the cell) or TIRF (movement at the cell surface and exocytosis) imaging - cells that do not have crowded FITC dextran labeled lysosomes are ideal for imaging the movement and exocytosis of individual lysosomes.</li>
	<li>Aligning the TIRF lasers for imaging microscope: Use 60X or 100X objective with &#62;1.45 NA. Set up the angle for incident TIRF laser beam using the manufacturer&#39;s approach.</li>
	<li>Injure the cell membrane by irradiating a small (1-2 mm2) region for &#60;100 msec, with the pulsed laser at 40-50% attenuation.</li>
	<li>To monitor the response of lysosome to cell injury, image cells at 4-6 frames/sec for at least 2 min or longer depending on the dynamics of exocytosis in the cells of interest.</li>
</ol>

<ol><li>Bi, G. Q., Alderton, J. M., Steinhardt, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium-regulated+exocytosis+is+required+for+cell+membrane+resealing%5BTitle%5D)+AND+%22Cell+Biol%22%5BJournal%5D)">Calcium-regulated exocytosis is required for cell membrane resealing.</a> Cell Biol. 131, 1747-1758 (1995).</li>
<li>Togo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disruption+of+the+plasma+membrane+stimulates+rearrangement+of+microtubules+and+lipid+traffic+toward+the+wound+site%5BTitle%5D)+AND+%22Cell+Sci%22%5BJournal%5D)">Disruption of the plasma membrane stimulates rearrangement of microtubules and lipid traffic toward the wound site.</a> Cell Sci. 119, 2780-2786 (2006).</li>
<li>Miyake, K., McNeil, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vesicle+accumulation+and+exocytosis+at+sites+of+plasma+membrane+disruption%5BTitle%5D)+AND+%22Cell+Biol%22%5BJournal%5D)">Vesicle accumulation and exocytosis at sites of plasma membrane disruption.</a> Cell Biol. 131, 1737-1745 (1995).</li>
<li>Reddy, A., Caler, E. V., Andrews, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plasma+membrane+repair+is+mediated+by+Ca2%2B-regulated+exocytosis+of+lysosomes%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Plasma membrane repair is mediated by Ca2+-regulated exocytosis of lysosomes.</a> Cell. 106, 157-169 (2001).</li>
<li>Babiychuk, E. B., Monastyrskaya, K., Potez, S., Draeger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blebbing+confers+resistance+against+cell+lysis%5BTitle%5D)+AND+%22Death+Differ%22%5BJournal%5D)">Blebbing confers resistance against cell lysis.</a> Death Differ. 18, 80-89 Forthcoming.</li>
<li>Bansal, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defective+membrane+repair+in+dysferlin-deficient+muscular+dystrophy%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Defective membrane repair in dysferlin-deficient muscular dystrophy.</a> Nature. 423, 168-172 (2003).</li>
<li>Abreu-Blanco, M. T., Verboon, J. M., Parkhurst, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+wound+repair+in+Drosophila+occurs+through+three+distinct+phases+of+membrane+and+cytoskeletal+remodeling%5BTitle%5D)+AND+%22Cell+Biol%22%5BJournal%5D)">Cell wound repair in Drosophila occurs through three distinct phases of membrane and cytoskeletal remodeling.</a> Cell Biol. 193, 455-464 (2011).</li>
<li>Jaiswal, J. K., Chakrabarti, S., Andrews, N. W., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synaptotagmin+VII+restricts+fusion+pore+expansion+during+lysosomal+exocytosis%5BTitle%5D)+AND+%22PLoS+Biol%22%5BJournal%5D)">Synaptotagmin VII restricts fusion pore expansion during lysosomal exocytosis.</a> PLoS Biol. 2, 1224-1232 (2004).</li>
<li>McNeil, P. L., Clarke, M. F., Miyake, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+wound+assays%5BTitle%5D)+AND+%22Protoc.+Cell+Biol%22%5BJournal%5D)">Cell wound assays.</a> Protoc. Cell Biol. Chapter 12, (2001).</li>
<li>McNeil, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+introduction+of+molecules+into+cells%5BTitle%5D)+AND+%22Protoc.+Cell+Biol%22%5BJournal%5D)">Direct introduction of molecules into cells.</a> Protoc. Cell Biol. Chapter 20, (2001).</li>
<li>Knight, M. M., Roberts, S. R., Lee, D. A., Bader, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+cell+imaging+using+confocal+microscopy+induces+intracellular+calcium+transients+and+cell+death%5BTitle%5D)+AND+%22J.+Physiol.+Cell+Physiol%22%5BJournal%5D)">Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death.</a> J. Physiol. Cell Physiol. 284, C1083-C1089 (2003).</li>
<li>McNeil, P. L., Miyake, K., Vogel, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+endomembrane+requirement+for+cell+surface+repair%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">The endomembrane requirement for cell surface repair.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 4592-4597 (2003).</li>
<li>Benink, H. A., Bement, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Concentric+zones+of+active+RhoA+and+Cdc42+around+single+cell+wounds%5BTitle%5D)+AND+%22Cell+Biol%22%5BJournal%5D)">Concentric zones of active RhoA and Cdc42 around single cell wounds.</a> Cell Biol. 168, 429-439 (2005).</li>
<li>Schmoranzer, J., Goulian, M., Axelrod, D., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+constitutive+exocytosis+with+total+internal+reflection+fluorescence+microscopy%5BTitle%5D)+AND+%22Cell+Biol%22%5BJournal%5D)">Imaging constitutive exocytosis with total internal reflection fluorescence microscopy.</a> Cell Biol. 149, 23-32 (2000).</li>
<li>Steyer, J. A., Horstmann, H., Transport Almers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((docking+and+exocytosis+of+single+secretory+granules+in+live+chromaffin+cells%5BTitle%5D)+AND+%22%22%5BJournal%5D)">docking and exocytosis of single secretory granules in live chromaffin cells.</a> 388, 474-478 (1997).</li>
<li>Jaiswal, J. K., Andrews, N. W., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane+proximal+lysosomes+are+the+major+vesicles+responsible+for+calcium-dependent+exocytosis+in+nonsecretory+cells%5BTitle%5D)+AND+%22Cell+Biol%22%5BJournal%5D)">Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells.</a> Cell Biol. 159, 625-635 (2002).</li>
<li>Jaiswal, J. K., Simon, S. M. Ch. 4.12. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aJaiswal%2C+JK+%22Current+Protocols+in+Cell+Biology%22">Current Protocols in Cell Biology</a>. , Wiley and Sons Inc. 4.12.1-4.12.15 (2003).</li>
<li>Johnson, D. S., Jaiswal, J. K., Simon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Total+internal+reflection+fluorescence+%28TIRF%29+microscopy+illuminator+for+improved+imaging+of+cell+surface+events%5BTitle%5D)+AND+%22Protoc.+Cytom%22%5BJournal%5D)">Total internal reflection fluorescence (TIRF) microscopy illuminator for improved imaging of cell surface events.</a> Protoc. Cytom. Chapter 12, (2012).</li>
<li>Jaiswal, J. K., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+single+events+at+the+cell+membrane%5BTitle%5D)+AND+%22Chem.+Biol%22%5BJournal%5D)">Imaging single events at the cell membrane.</a> Chem. Biol. 3, 92-98 (2007).</li>
</ol>

The authors have nothing to disclose.

Cell membrane injury in vivo occurs due to a variety of physiological stressors and several experimental approaches have been developed to mimic these. These include injuring cell membrane of adherent cells by scraping them off the dish or by passaging through a narrow bore syringe9,10. Following such injuries the cells heal in suspension and not adhered to the extracellular matrix as they normally do in the tissue. Still others, such as use of pore forming toxins chemically alter cell membrane by ext...

Cell membrane maintains the integrity of cells by providing a barrier between the cell and the extracellular environment. A chemical, electrical, or mechanical stimulus that exceeds the normal physiological threshold as well as the presence of invading pathogens can each result in injury to the cell membrane and trigger a subsequent cellular response to repair this injury. To survive these injuries to the cell membrane, cells possess an efficient mechanism for repair. This mechanism is calcium dependent and involves intracellular trafficking of proteins such as annexins and MG53 amongst others as well as subcellular compartments such as endosomes, lysosomes, Golgi derived vesicles and mitochondria to the injured cell membrane1-7. However, the details of the sequence of molecular and subcellular events involved in repairing damaged cell membrane remains poorly understood.
Cell&#39;s repair response can be segregated into early and late responses. Early responses, which occur within seconds to minutes time scale, are tremendously important in determining the nature of late responses leading to successful cell repair or cell death. End point assays based on bulk biochemical and cellular analysis have helped establish the involvement of molecular and cellular processes in repair. But, due to the heterogeneity and rapidity of cellular repair response, end point assays fail to provide the kinetic and spatial details of the sequence of events leading to repair. Approaches that enable controlled injury of cell membrane and allow monitoring the cell membrane repair and associated subcellular responses at high spatial and temporal resolution are ideally suited for such studies. Here, such approaches have been presented. Two of the protocols describe approaches to monitor the real time kinetics of cell membrane repair and the subcellular responses associated with the repair process in live cells following laser injury. As a complement to these live cell imaging based assays, end point assays have also been described that provide a population based measure for monitoring repair of individual cells and the associated subcellular responses. To demonstrate their utility these approaches have been used to monitor trafficking and exocytosis of lysosomes in response to cell membrane injury.

<table><tbody><tr><td>HBSS</td><td>Sigma</td><td>H1387</td><td>Used to prepare CIM (HBSS/10 mM Hepes/2 mM Ca&lt;sup&gt;2+&lt;/sup&gt; pH 7.4)</td></tr><tr><td>HEPES</td><td>Fisher</td><td>BP410</td><td>Used to prepare CIM (HBSS/10 mM Hepes/2 mM Ca&lt;sup&gt;2+&lt;/sup&gt; pH 7.4)</td></tr><tr><td>FITC dextran (Lysine fixable)</td><td>Invitrogen</td><td>D1820</td><td>2 mg/ml in CIM or PBS</td></tr><tr><td>Glass beads</td><td>Sigma</td><td>G8772</td><td></td></tr><tr><td>TRITC dextran (Lysine fixable)</td><td>Invitrogen</td><td>D1818</td><td>2 mg/ml in CIM or PBS</td></tr><tr><td>PFA 16%</td><td>EMS</td><td>15710</td><td>4% in PBS</td></tr><tr><td>Hoechst</td><td>Invitrogen</td><td>H3570</td><td>1/10 000 in PBS</td></tr><tr><td>FM dye</td><td>Invitrogen</td><td>T3163</td><td>1 &amp;micro;g/&amp;micro;l in CIM or PBS</td></tr><tr><td>FITC dextran</td><td>Sigma</td><td>FD40S</td><td>2 mg/ml in growth medium</td></tr><tr><td>LAMP1&amp;nbsp;</td><td>Santa Cruz</td><td>sc-19992</td><td>1/300 in growth medium</td></tr><tr><td>Alexa Fluor 488 chicken anti-rat IgG</td><td>Invitrogen</td><td>A21470</td><td>1/500 in blocking solution</td></tr><tr><td>Mounting media</td><td>Dako</td><td>S3023</td><td></td></tr><tr><td>Coverslips</td><td>Fisher</td><td>12-545-86</td><td></td></tr><tr><td>Glass bottom petridish</td><td>MatTek corporation</td><td>P35G-1.0-14-C</td><td></td></tr><tr><td>Silicone O ring</td><td>Bellco</td><td>1943-33315</td><td></td></tr><tr><td>Coverslip holder</td><td>Bellco</td><td>1943-11111</td><td></td></tr><tr><td>Invitrogen</td><td>A-7816</td><td></td><td></td></tr><tr><td>DMEM</td><td>VWR</td><td>12001-566</td><td></td></tr><tr><td>FBS</td><td>VWR</td><td>MP1400-500H</td><td>Used for growth medium: 10% FBS, 1% P/S in DMEM</td></tr><tr><td>Penicillin/Sreptomycin</td><td>VWR</td><td>12001-350</td><td>Used for growth medium: 10% FBS, 1% P/S in DMEM</td></tr><tr><td>Chicken serum</td><td>Sigma</td><td>C5405</td><td>5% chicken serum in CIM is used for blocking solution during immunostaining</td></tr><tr><td>PBS (Ca2+ and Mg2+ free)</td><td>VWR</td><td>12001-664</td><td></td></tr><tr><td>Epifluorescence microscope</td><td>Olympus America, PA</td><td>IX81</td><td></td></tr><tr><td>TIRF illuminator&amp;nbsp;</td><td>Olympus America, PA</td><td>Cell^TIRF (IEC60825-1:2007)</td><td></td></tr><tr><td>Epifluorescence illuminator</td><td>Sutter Instruments, Novato CA</td><td>Lambda DG-4 (DG-4 30)</td><td></td></tr><tr><td>CCD Camera</td><td>Photometrics, Tucson, AZ</td><td>Evolve 512 EMCCD&amp;nbsp;</td><td></td></tr><tr><td>Image acquisition and anaysis software</td><td>Intelligent Imaging Innovations, Inc. Denver, CO</td><td>Slidebook 5</td><td></td></tr><tr><td>Pulsed laser</td><td>Intelligent Imaging Innovations, Inc. Denver, CO</td><td>Ablate (3iL13)</td><td></td></tr><tr><td>Stage top incubator</td><td>Tokaihit, Japan</td><td>INUBG2ASFP-ZILCS</td><td></td></tr></tbody></table>

imaging cell membrane injury and subcellular processes involved in repair

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

Protocols described here for single cell imaging are to monitor the ability and kinetics of cell membrane repair (Protocols 1 and 2) and the subcellular trafficking and fusion of lysosomes during repair (Protocols 3 and 4).
Protocol 1 shows a bulk assay that allows marking all injured cells and identifying those injured cells that failed to repair. The results in Figure 1 show that while the uninjured cells (Figure 1A) remain unlabeled, cells injured by glass ...

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

The overall goal of the following experiment is to observe the effect of desired treatments on repair of injured cells and to study the subcellular processes that help in repairing injured cells. This is achieved by injuring cells in the presence of fluorescent dyes that cannot cross intact cell membranes to assess the kinetics and ability of injured cells to prevent entry of these dyes. As a second step, we monitor the appearance of intracellular proteins at the injured cell membrane, which identifies the ability of a cellular compartment of interest to participate in repair by fusing to the injured cell membrane.Next, we monitor fusion of cellular compartments of interest with the injured cell membrane in order to monitor the kinetics of injury. Triggered fusion of this compartment results are obtained that show repair of cell membrane injury is associated with lysosomal exocytosis based on increased appearance of lysosome associated membrane protein, one at the injured cell surface and live imaging of secretion of lysosomal content by injured cells. These methods can help address important questions in cell injury and repair.For example, these can be used to determine which proteins and cellular compartments respond to focal injuries at the cell membrane and then evaluate their kinetics of the response. Demonstrating the procedure would be Aurelia D four and hamma stra two of the postdoctoral fellows from my laboratory. To separately mark the injured cells that heal and those that fail to heal.Seed the cells on sterile cover slips for the three experimental conditions. Culture the cells till they reach greater than 50%confluence, then wash C one and C two twice with CIM at 37 degrees Celsius. Wash C3 with PBS at 37 degrees Celsius.Then transfer the cover slips on silicone O-rings.Rings. Add 100 microliters of prewarm fitzy dextran solution in appropriate buffer. Injure the plasma membrane on C one and C3 by adding 40 milligrams of glass beads over the cover slip at room temperature and manually tilting the cover.Slips back and forth six to eight times at an angle of 30 degrees for mild injury. Ensure the glass beads are spread uniformly, thus minimizing repeat injuries to improve reproducibility of injury between samples. Simultaneously carry out the glass bead injury of the different samples to be compared, avoiding further rolling of beads.Transfer all cover slips to a 37 degrees Celsius incubator at ambient CO2 and allow repair to proceed for five minutes without letting the beads roll. Remove the glass beads and fitzy dextran by washing with buffer or PBS. Now place the cover slips back on the O-ring and add 100 microliters of prewarm lysine.Fixable trissy dextran solution Incubate at 37 degrees Celsius for five minutes at ambient CO2. Wash the cover slips twice with prewarm CIM and fix with 4%PFA for 10 minutes at room temperature after two PBS washes. Incubate for two minutes in hersch dye and after two PBS washes mount the sample on a slide using mounting media.Next, image the cells using an epi fluorescence microscope from the C two sample. Determine the background red and green staining intensity. Then use this value to threshold the red and green channels for all samples.Score the total number of cells that are green. Also score cells that are red or both red and green in the C one and C3 samples. Now count over 100 green injured cells for each condition and express the fraction of cells that failed to repair as a percent of all cells injured.Wash the cells grown to over 50%Confluence with pre-warned CIM and mount the cover slip in CIM with FM dye. In a holder in the stage top incubator maintained at 37 degrees Celsius. Select a region of the cell membrane using optimal power that allows consistent but non-lethal injury.Irradiate the sample for less than 10 milliseconds with the pulsed laser to achieve this. Attenuate the laser power through the software to 40 to 50%of the peak power in order to monitor, repair, acquire images every two to 10 seconds in epi, fluorescence and brightfield starting prior to injury and continuing for three to five minutes following injury. To quantify the kinetics of repair, measure cellular FM dye fluorescence.Calculate the change in intensity during the course of imaging. Average the data for over 10 cells in each condition and plot as either averaged or individual cells value. Wash the cover slips of C one and C two.Culture preparations with CIM at 37 degrees Celsius and wash C3 with PBS at 37 degrees Celsius. Then transfer the samples on silicone O-rings. Add 100 microliters of prewarm lysine fixable trissy dextran.Injure the plasma membrane on C one and C3 as shown earlier. Then avoiding further rolling of beads. Transfer all cover slips to a 37 degrees Celsius incubator at ambient CO2 and allow repair to proceed for five minutes.Now to remove the glass beads and Trixie dextran wash the cover slips in cold growth medium, ensuring no rolling of the glass beads on the cells. Rinse the cover slips twice with cold growth medium and transfer to the O-rings to C one and C3.Add 100 microliters of rat anti-US lamp one antibody in cold complete growth media and add the cold complete growth media to C two to allow antibody binding. Incubate cover slips for 30 minutes at four degrees Celsius after three washes with cold CIM.Fix all cover slips with 4%PFA for 10 minutes at room temperature. Then rinse thrice with CIM. Next, incubate all the cover slips in 100 microliters a blocking solution for 15 minutes, followed by 100 microliters ofor, 4 88 anti rat antibody for 15 minutes.Wash twice with PBS and incubate in her stain for two minutes at room temperature after two PBS washes. Mount the samples on a slide, then image the samples using an epi fluorescence microscope using images of C two cells. Determine the nonspecific background staining in the red and green channels.Use these background staining values to threshold the red and green channels for all samples. Use the over 100 red labeled cells from C one and C3 to measure the intensity of lamp one staining in injured cells. In a successful experiment, the lamp one staining cells from C3 will be significantly lower than in cells from C one for labeling lysosomes with fluorescent dye incubate the cells grown to 50%confluence in growth media containing fitzy dextran.Allow the dextran to be endocytose for two hours in the cell culture incubator. Wash the cells with Prewarm growth media and incubate in it for two hours in a CO2 incubator to allow all endocytose dextran to accumulate in the lysosome before imaging. Rinse the cover slip in Prewarm CIM and mount in CIM on a cover slip holder on the stage top incubator at 37 degrees Celsius.Select for cells that do not have crowded fitzy dextran labeled lysosomes for imaging. The movement and exocytosis of individual lysosomes. Acquire wide field images for lysosome movement throughout the cell.Alternately acquire turf images for movement at the cell surface and to monitor their exocytosis. Injure the cell membrane by irradiation as shown earlier to monitor the response of lysosome to cell injury image cells at four to six frames per second for at least two minutes or longer depending on the dynamics of exocytosis in the cells of interest. In this bulk assay, all the injured cells are marked and those that failed to repair are identified while the uninjured cells remain unlabeled.The cells injured by glass beads in the presence of fitzy dextran are labeled green when the cells are allowed to repair in the presence of calcium. Most of the injured cells manage to repair and are not marked by trissy dextran when the cells are allowed to repair. In the absence of calcium, most of the injured cells are also labeled red by the trissy dextran.This experiment assesses the kinetics of cell membrane repair by monitoring the entry of FM dye in the cell. When incubated in FM die intact cells show little to no intracellular staining. Following localized laser injury of the cell membrane, FM D starts entering the cell and binding endo membranes, which causes a sudden increase in FM dye fluorescence as the capacity of the cell membrane to repair following laser injury is dependent on calcium.A cell injured in the presence of calcium is able to repair, which causes the FM dye entry to cease within a minute after injury. On the contrary, a cell allowed to repair in the absence of calcium fails to repair, which results in continuous dye entry and hence continuous increase in FM d fluorescence even four minutes after injury. Thus, cell membrane repair leads to a plateauing of the FM dye staining.While lack of cell membrane repair causes continuous dye entry that fails to plateau. The bulk glass bead injury approach can be used to monitor cell surface translocation of vesicles and proteins in response to injury. Here, the cells are injured in the presence of trissy dextran.Thus, while the uninjured cells are not labeled red, all the injured cells are labeled red. Note that uninjured cells that are not treated with primary antibody show background level labeling for cell surface lamp one. However, when cells are injured and allowed to heal in the presence of calcium, lysosomes undergo exocytosis and thus there is an increased level of lamp one staining on the surface of the injured cells.This increase in cell surface lamp one labeling is much lower in cells that are allowed to heal in the absence of calcium. Thus both quantifying the number of cells with high cell surface lamp one staining, as well as measuring the level of cell surface lamp.One. Staining on individual injured cells provide measures for the cell's ability to undergo injury triggered lysosomal exocytosis.This experiment directly monitors the kinetics, nature and location of individual lysosome fusion. In response to cell membrane injury, the cells are injured by the pulsed laser and cell surface lysosomes are imaged by turf imaging, which allows monitoring injury triggered exocytosis of lysosomes in individual cells. This figure shows a cell that is visualized by phase contrast imaging in the left panel and by turf microscopy in the middle panel.The right panel shows the turf image of the same cell 105 seconds after injury. Here, injury triggered recruitment of a lysosome to the cell membrane, followed by exocytosis is shown. This lysosome is not visible in the turf image prior to injury, but following injury, the lysosome arrives at the cell membrane and becomes detectable.As the vesicle moves closer to the cell membrane, it is better excited by the turf illumination causing the lysosomal fitsy dex strand fluorescence to reach maximal value. When the fusion pour opens, this causes neutralization of the lysosomal pH and thus de quenching of the fitzy DExT strand fluorescence. Subsequently, the DExT strand is discharged from the vesicle to the exterior of the cell, causing ZI fluorescence to spread laterally and the vesicle fluorescence to gradually decrease various injury triggered responses of the lysosomes can be measured.After watching this video, you should have a good understanding of how to monitor the ability of cells to repair from large focal injuries and to monitor subcellular compartments and proteins that respond to and help with the repair process. Further experiment using different tag vital labeling, compartments or protein of can be performed in order to monitor the response to injury and the involvement in the repair of IND cells.

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Research

JoVE Journal

Biology

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

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Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

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Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

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.

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

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.

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

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

The thermoplasmonic puncture method integrates confocal microscopy, optical tweezers, and gold nanoparticles to study protein responses during plasma membrane repair in cells and giant unilamellar vesicles. The technique enables rapid and localized membrane puncture, allowing the identification of key proteins and their functional roles in the intricate plasma membrane repair machinery.

A Thermoplasmonic Approach for Investigating Plasma Membrane Repair in Living Cells and Model Membranes

Learn to image cell membrane injury and repair using fluorescent dyes and live microscopy. This protocol reveals lysosomal exocytosis dynamics and subcellular mechanisms that enable injured cells to restore membrane integrity.

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

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