The overall goal of this procedure is to quantify plasma membrane repairability in muscular dystrophy patient cells. This method can help answer key questions in muscular dystrophy, such as what is the relationship between a given mutation and membrane resealing kinetics. The main advantage of this technique is that it allows realtime quantification of membrane resealing kinetics in living cells.
Begin by culturing fibroblast cells in a T225 flask containing 40 milliliters of growth medium in a carbon dioxide incubator at 37 degrees Celsius. When the culture reaches 70 to 80%confluency, rinse the cells two times with 40 milliliters of PBS per wash, followed by the addition of five milliliters of 0.05%trypsin for five minutes at 37 degrees Celsius. When the cells have detached, stop the reaction with 40 milliliters of fresh growth medium and seed two milliliters of cells at a 100, 000 cells-per-milliliter concentration into 35-millimeter collagen-coated glass-bottom plates for an overnight incubation at 37 degrees Celsius and 5%carbon dioxide.
Add 150 microliters of fresh serum-deprived medium to one 1.5-milliliter tube per transfection culture. Next add three microliters of transfection reagent to each tube, and incubate the tubes for at least five minutes at room temperature. While the tubes are incubating, add 175 microliters of serum-derived medium and 3.5 micrograms of dysferlin plasmid DNA to an additional 1.5-milliliter tube per plate.
Then combine 150 microliters of the plasmid DNA solution with 150 microliters of the transfection reagent into one new tube per plate for a 20-minute incubation at room temperature. The next morning, replace the growth medium with two milliliters of serum-derived medium. At the end of the incubation, evenly distribute the entire 300 microliters of plasmid solution across each plate, and incubate the plates for 24 hours in the carbon dioxide incubator at 37 degrees Celsius.
The next day, replace the plasmid supernatant with fresh growth medium and return the plates to the incubator for another 24 hours. To prepare the cells for a two-photon wounding assay, rinse the transfected cultures one time with one milliliter of Tyrode's solution. Then add one milliliter of fresh Tyrode's solution supplemented with one microliter of 2.5-millimolar FM4-64 dye to the cells.
Next, using an inverted confocal microscope, create a 0.2 by two micron target in the microscope's software, and place the target at the edge of the cell membrane so that the target line overlaps and lies perpendicular to the cell membrane. Select a 543-nanometer helium neon laser to excite the FM dye signal, and a 488-nanometer argon laser to excite the GFP signal, and set the detection range for 600 to 760 nanometers and 500 to 550 nanometers respectively. Then create a five-minute time series of sequential image scans.
To generate a membrane lesion, use a two-photon laser to 820 nanometers at 15%laser power with 10 iterations to bleach the cells 25 seconds after the beginning of the time series. To quantify the FM4-64 dye fluorescence, use the software to draw a six by six micron region of interest, and place the region of interest adjacent to the wounding location within the cell. Then subtract the background value and divide the net increase by the fluorescence value at time zero to calculate the relative fluorescence values for each time point.
Healthy human fibroblast cells display low levels of FM4-64 fluorescence activation following laser wounding while non-treated patient fibroblasts exhibit a high degree of relative fluorescence intensity following injury. Patient cells that were transfected with full-length dysferlin plasmid demonstrate a reduced relative FM4-64 fluorescence intensity compared to non-treated patient controls but similar to the fluorescence values observed in healthy controls. While attempting this procedure, it's important to remember the cells will slowly die in the buffer solution, so work quickly and change to fresh cells at regular intervals.
After watching this video, you should have a good understanding of how to regenerate the membrane lesions using a two-photon microscope and how to quantify membrane resealing in realtime.
