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.
