This demonstration aims to image fluorescently tagged proteins in live cells in A 3D matrix. First, generate stable cell lines expressing fluorescently tagged proteins. Seed these cells into a 3D collagen matrix, then acquire time-lapse images of migrating cells using a confocal microscope.
Ultimately, results illuminate protein localization and dynamics within migrating cells through time-lapse imaging and FP analysis, Observing protein dynamics and localization in 3D and in real time. Offers one avenue for addressing fundamental questions in cell migration. For example, how are adhesive contacts regulated by cytoplasmic proteins?
And also how are these contacts perturbed by specific inhibitors. In a P 35 dish culture cells at 80 to 90%co fluency transfect the cells with the plasmid of interest using lipo 2000 as per manufacturers instructions. Then place the cells in an incubator.
On the next day, split the cells into two P one 50 Petri dishes and incubate overnight. Replenish the media to add 50 micrograms per milliliter of G four 18. Continue culture under antibiotic selection for two weeks.
Examine the culture for G four 18 resistant colonies. Then using an inverted fluorescent microscope. Identify and mark GFP positive colonies.
Wash the cells twice with PBS. After the second wash, leave a thin layer of liquid to prevent cells from drying for each marked colony. Wipe as close as possible around the edge with a sterilized cotton swab and pipette 10 microliters of trypsin onto the colony.
Repeat swiftly for every colony. Then incubate the plate at 37 degrees centigrade for cell detachment. Verify round appearance under a microscope.
Add 10 microliters of trypsin onto each colony, pipette to fully detach cells from the plate. Then transfer each colony of cells into a single well of a 24 well dish culture to amplify stable colonies. Next, evaluate protein expression of the colonies using standard western blot and immunofluorescence.
Expand selected cell lines surface modification of the glass. Bottom dish affords optimal collagen binding. Pipette 300 microliters of siloization solution onto the glass portion of each P 35 dish with a 10 millimeter opening.
Incubate for one hour at room temperature. Aspirate out the solution and wash with filtered water three times for 10 minutes each. Remove the water and place dishes on a 50 degrees centigrade hot plate for 1.5 hours.
Position tops of dishes slightly off the dish to permit drying. After the dry dishes have cooled pipette 300 microliters of 2%glutaraldehyde solution onto the glass portion of each dish. Incubate for one hour, then wash with PB S3 times for 10 minutes each.
Proceed to sterilize dishes by exposure to UV light for one hour. First pellet 100 microliters of fluorescent tracer particles by centrifugation. Discard the liquid and resuspend particles in 500 microliters of media.
Perform five washes, then resuspend the particles in 30 microliters of media. Next, harvest the GFP expressing cells using trypsin and prepare a suspension of 2 million cells per milliliter into an einor tube Pipette 240 microliters of growth media. Add 12.6 microliters of one molar heaps, 20 microliters of filtered water, 50 microliters of cell solution, and 10 microliters of the fluorescent particles.
Lastly, add 167 microliters of bovine dermis collagen, one solution mix solution thoroughly. Now transfer 80 microliters onto the glass portion of the prepared siloized dish. Incubate at 37 degrees centigrade for 50 minutes for the gel to polymerize.
Then carefully add two milliliters of growth media. Equilibrate the microscope chamber to a steady state temperature of 37 degrees centigrade replenish cell medium. To maintain a neutral pH for DIC imaging exchange the top of the dish to a glass top with a thin strip of paraform.
Cover the side of the dish to prevent evaporation for an oil immersion objective. Place one drop of immersion fluid on the objective position. The collagen gel containing the dish on the microscope stage so the dish makes contact with the immersion liquid.
Focus the sample and search for cells of interest to image to minimize stage drift. Allow dish to settle for about 45 minutes. Before starting a long capture, specify the parameters of image acquisition for inhibitor addition experiments.
Prepare supplemented media with drug at a desired working concentration. Replenish cell medium to maintain a neutral pH, but do not seal with perfil. Transfer the sample to the microscope and proceed with imaging at the time of drug treatment.
Pause the image, capture and carefully remove the dish top without disturbing the dish. Aspirate the media and pipette the drug containing media into the dish. Then carefully replace the dish.
Top frap setup varies between systems. So do consult manufacturer's instruction. First set parameters for live cell imaging.
For photobleaching. Test these parameters on practice cells to obtain sufficient laser power to photobleach the fluorescent signal without damaging the cell. Next, start image capture on sample acquiring at least five frames.
Then photobleach the region of interest. Continue to capture over the recovery time. Measure the average fluorescent intensity of the photo bleached area before and after photobleaching over time, using a statistical analysis software.
Fit the recovery curve to the exponential equation. Obtain the parameters for halftime and final intensity from the exponential fit curve. Then calculate the mobile fraction by taking the ratio of the final to initial fluorescence intensities.
These 3D live cell images show healthy epithelial cells expressing GFP actin. After four days of culture, these cells formed a cyst in a 3D collagen matrix. Some cells migrated along the surface of the cyst while others migrated within the interior of the cyst.
Matrix deformation as a result of the traction forces exerted by migrating cells, is analyzed through the displacement of tracer particles embedded in the surrounding matrix. The effect of RO kinase inhibition on traction force is shown here. The tracer particle movements are displayed as a maximum projection image of different time points and each time point is pseudo colored.
Alternatively, the images of an individual tracer particle are displayed as a montage to show the movement of the tracer particle. Over time, this tracer particle moved toward and away from the trailing edge of the migrating cell before and after the addition of Y 2 7 6 3 2 respectively. The frap analysis follows cells expressing GFP actin as they migrate in a three-dimensional matrix indicating typical fluorescence recovery After a photo bleaching experiment in optimized laser settings, fluorescence intensity of the region of interest is visibly diminished compared to the background level while maintaining healthy and undamaged cells.
This graph shows the fit of the recovery curve with an exponential function. Once mastered, seeding the cells in the matrix can take an hour. Don't forget that working with three amino propyl trimethyl can be extremely hazardous.
So take precautions like working in the chemical hood. Also remember to maintain sterile conditions when working with cells in a 3D matrix.
