Live-Cell Imaging of Single-Cell Arrays called LISCA are also an automated readout of the fluorescent time courses of hundreds of individual cells. The advantage of the approach is that individual cell responses are recorded from spatially isolated cells in identical micro environments, allowing an efficient emission analysis with great statistics. To fabricate a single-cell array, use a scalpel to cut a single PDMs masterpiece containing six micro powder stripes out of the PDMs layer, and place the masterpiece on the lab bench with the micro pattern facing up.
Use a razor blade to cut each of the six micropattern stripes into a PDMs stamp, cutting off some of the patterned areas to ensure that the edges of the stamp are open. Next, carefully scratch the protective foil of the cover slip of a six chamber slide to mark the channel positions of the slide and place the cover slip on the bench with the protective foil facing down. Use tweezers to place the stamps on the cover slip at the marked channel positions, with the micro patterns facing down, and check the attachment of the stamps under a microscope.
The squares in contact with the slide will appear darker than the interspace. The pattern quality is decreased by squares that are not properly attached to the slide. If the stamps have securely attached treat the cover slip and the stamps with oxygen plasma at 0.2 millibars of pressure and approximately 40 Watts for three minutes to make the surfaces between the PDMs stamps and the cover slip hydrophilic.
At the end of the plasma cleaning, place the cover slip into a biosafety cabinet and add 15 microliters of pegylated poly-l-lysine solution to each stamp so that the solution is absorbed into the hydrophilic pattern of the stamp. After 20 minutes, rinse the stamps with one milliliter of ultrapure water. Use tweezers to remove the stamps from the slide and rinse the cover slip a second time with a second milliliter of ultrapure water.
When the cover slip has dried completely, attach a six channel sticky slide to the cover slip taking care that the micro patterned areas align with the bottom of the channels. And add 40 microliters of PBS and 40 microliters of fibronectin solution to each channel. To mix the two solutions, remove 40 microliters from one reservoir and add it to the opposite reservoir of the same channel three times to generate a homogenous solution.
When all of the channels have been mixed, incubate the slide for 45 minutes at room temperature before washing each channel three times with 120 microliters of PBS per wash. Exchange the PBS to 37 degrees Celsius, fully supplemented cell growth medium in the channels by adding 120 microliters medium to one reservoir and remove it from the opposite reservoir. Add 40 microliters of a 400, 000 cells per milliliter cells suspension of interest and 40 microliters of cell growth medium to each channel, and mix the cell solution with the medium as demonstrated.
After mixing, remove 40 microliters of suspension from each channel so that only the channels are filled with cell suspension, and place the slide into a cell culture incubator. After one hour, check for cell adhesion by phase contrast microscopy, and add 120 microliters of 37 degrees Celsius cell growth medium to each channel, then return the slide to the cell culture incubator for three more hours. To set up a perfusion system, connect a one milliliter syringe filled with 37 degrees Celsius cell growth medium to the inlet tube of the system, and use the valve to fill the tube with medium.
Connect the inlet tube to the reservoir of one channel, taking care that no air bubbles are trapped, and connect a serial connector to the reservoir opposite to the inlet tube of the current channel. Connect the rest of the channels as demonstrated until the required number of channels are connected in series. And connect the outlet tube directly to the free reservoir of the final channel.
Then fill the connected tube with medium to confirm that the perfusion system does not leak. For time-lapse imaging of the cells, to set up a time lapse protocol for recording a phase contrast image and a fluorescence image, set the optical configuration for phase contrast imaging and fluorescence imaging. Use a 10 times objective, the appropriate fluorescence filters and an automated focus correction system to ensure a better image quality for the longterm measurements.
Select a 10 millisecond exposure time for the phase contrast imaging and a 750 millisecond exposure time for recording the EGFP fluorescence intensity. Set a time schedule with a 10-minute time interval between the consecutive loops through the position list and an observation time of 30 hours. Next, place the six channels slide on the single cellar raise in the sample holder of the 37 degrees Celsius heating chamber of the microscope and tape the perfusion system tubing to the stage.
Insert the free ends of the outlet tubes into a 15 milliliter conical tube to collect the liquid waste. And set the position list for the scanning time lapse measurement, taking care that the number of positions will be able to be scanned within the defined time interval between consecutive loops through the position list. Then start the time lapse measurement.
For transfection of the cells with a fluorescent marker, use a syringe to flush the tubing system with one milliliter of 37 degrees Celsius PBS, taking care that the microscope stage doesn't move. After flushing, fill the system with 300 microliters of serum reduced medium supplemented with the mRNA of interest, to a final concentration of 0.5 nanograms of mRNA per microliter, and allow the lipoplexes to incubate for one hour. At the end of the incubation, flush the system with one milliliter of 37 degrees Celsius complete cell growth medium to remove any unbound mRNA.
At the end of the analysis, view the channels listed in the channel menu and scroll through the timeframes to inspect that preselected cells and their integrated fluorescent signals. Click on a cell of interest to highlight its fluorescence time course, or click on a time course to find the corresponding cell. Press shift while clicking on a cell to select or de-select it.
De-select any cells that are not viable or not confined to an adhesion spot or that are attached to another cell to exclude them from further analysis. When all of the cells are selected or deselected appropriately, save the single cell time courses for the cell area and the integrated fluorescence in an appropriate directory. To quantify the translation onset time after the transfection, and the strength of the cellular EGFP expression, a three-stage translation model can be employed.
The model solution for EGFP can be fitted to single cell time courses as illustrated. Here, histograms of the translation onset time and the expression rate of lipoplex transfected cells can be observed. As both parameters are estimated for each cell, the correlation of these parameters can be analyzed as demonstrated in the scatter plot, and compared to cells transfected with lipid nanoparticles.
As illustrated, cells transfected with lipid nanoparticles exhibit less cell-to-cell variability compared to cells transfected with lipoplexes. And the population average demonstrates a faster onset of translation, as well as a higher expression rate. Our approach can also be adapted to alternative micro-patterning methods.
Most important for LISCA is a good cell confinement and a combination of automated image analysis with a convenient user supervision. As you have seen, cell behavior is heterogeneous. And single-cell time lapse movies provide access to unbiased dynamics of the inherent biochemical networks.
For example, in green expression, apoptosis or cell killing assays.
