The overall goal of this procedure is to create adhesive and soluble gradients to study cell migration. This is accomplished by first passivating, a surface to prevent nonspecific cell adhesion. The second step is to fabricate stamps from micro contact printing.
Next, the ated surface is patterned with strep avid lines by micro contact printing. Strapped din can also be printed as dots using dip pen lithography attached to the bottom of a microfluidic device. The modified surface will act as a foundation for an adhesive gradient of biotin RGD.
The final step is to load cells onto the surface with the adhesive cue in the presence of a soluble chemo attractant gradient. Ultimately, live cell microscopy is used to show cell migration in response to both adhesive and soluble cues. The main advantage of this technique over existing methods such as transwell or void chamber assay and a conventional microfluidic setup is that disintegrated system of surface pattern Microfluidic device allows the observation of cells responses to spatially controlled adhesive cues and adhesive gradient and a soluble gradient at the same time.
Well, this method can help answer key questions in the cell migration field, such as the importance of cell adhesion and cause stroke between chemo Texas receptors such as receptor seen kinase G-protein coupled receptors and adhesion receptors like RIN To ate glass cover slips. First, clean the cover slips by submerging them in a rack in 100%ethanol and placing the rack in a son skating water bath for 30 minutes. Dry the glass cover slips in air.
Next sonicate the glass cover slips for 30 minutes in one molar sodium hydroxide. Afterwards, rinse the glass surfaces by carefully tipping the rack in a beaker filled with one and a half liters of milli cube water. Repeat the rinsing process three times using fresh milli cube water each time.
After the third rinse dry the glass cover slips in an oven at 60 degrees Celsius for about half an hour. Place half of the clean glass cover slips individually in a humid chamber that is made of a 24 well plate, partially filled with water. Wells are filled with water and the glass cover slips are laid on top of the wells for the ation, A copolymer of polylysine and polyethylene glycol is used in which 20%of peg molecules are grafted to biotin.
Abbreviated as PLL Peg biotin drop 15 microliters of PLL Peg biotin in PBS on each glass cover slip. Take the other half of the remaining clean glass cover slips and carefully sandwich the PEG solution. Leave the cover slips for one hour in the humid chamber.
After an hour slide, the sandwiched cover slips gently away from each other without scraping the PEG coated surface and rinse them with UE water. The water should slide off easily on the peg treated side. If necessary.
Air dry the glass cover slips on a paper towel with the treated surface facing upward store. The glass cover slips in a vacuum desiccate until further use. The silicon master required for this procedure is fabricated by photolithography to cast A-P-D-M-S stamp for the PDMS elastomer onto the Silicon Master inside a Petri dish to about one centimeter in height and cure the master and PDMS mixture in an oven at 70 degrees Celsius for one hour.
Subsequently, the PDMS stamp is removed from the Silicon master and cut to size to print protein patterns using the micro contact printing technique first clean and to make the PDMS stamps more hydrophilic by treating the pattern side in a UV and ozone cleaner for one and a half hours immediately after the ozone treatment place. And spread 10 microliters of stripped adin onto the PDMS stamps. If tracks should be visible under the fluorescent microscope, use stripped Aden that is conjugated to a fluoro four, such as stripped avid and LOR three 50.
Leave for one hour in a humid chamber. Remove excess strep din from the stamp with tissue, paper and air. Dry the stamp for approximately one minute.
Lightly press the stamp onto the PLL peg biotin coated glass cover slip. If fluorescent strep din was used, examine the pattern under an epi fluorescence microscope. Protein patterns can also be printed using dip pen lithography.
First mix one part of one milligram per milliliter, strippin or stripped avidan elif Fluor three 50 with 10 parts of glycerol and load five microliters of the mixture onto the cantilever. Adjust printing speed. Contact time of the cantilever on the surface or a vertical distance of the cantilever to the surface to reduce the spot size.
Approximately five micrometers begin the printing process as described in the operation manual of the dip pen lithography instrument store. All printed surfaces in a desiccate. A commercially available microfluidic device is used to create surface gradients.
Adhere this tritin coated or patterned glass cover slips onto the sticky side of the device to ensure that there is no leakage for several hours. Seal the edges of the device with the thin layer of warmed Vaseline and with a second layer of a mixture of one part Vaseline to one part paraffin wax. Fill the channel of the device with six microliters of PBS to create two opposing gradients.
Fill the two reservoirs, one with 70 microliters of biotin, four fluorescein and the other with 70 microliters of biotin conjugated to biotinylated peptide arginine glycine spartic acid or RGD incubate the samples at room temperature in the dark for one hour. Rinsing biotin, RGD and biotin fitsy of the microfluidic device is the most difficult aspect of this procedure because there's the risk of trapping air bubbles and disturbing the micro contact printed tracks. To ensure success, the biotin and PBS must be removed carefully and slowly.
Remove the biotin solutions and carefully rinse the surface twice with PBS while still attached to the microfluidic device. Mark the direction of the adhesive gradient prior to loading fluorescently labeled cells into the microfluidics device. Count the cells and divide them into two tubes of equal cell numbers.
Wash and resus. Suspend one tube of cells with media containing the chemo attractant, such as low glucose DMEM with 10%FBS wash and the other tube of cells in media. Without the chemo attractant such as DMEM with 0%FBS, the final concentration of cells in each tube should be five times 10 of the fifth cells per milliliter.
Remove all the solutions from the microfluidic device and place it on the prewarm microscope. Stage load 70 microliters of cells resuspended in 0%F-B-S-D-M-E-M into one reservoir. Load 70 microliters of cells resuspended in 10%F-B-S-D-M-E-M into another reservoir.
Loosely placed tape over the reservoirs to avoid evaporation. Ensure that the channel of the microfluidic device is in the field of view and cells are in focus. Select image acquisition parameters such as exposure times and fluorescent filters.
Image sequence of brightfield and fluorescence that cells and tracks as well as time points and recording length start the image acquisition program. This video demonstrates a method for live cell imaging of cells that migrate in an environment with competing adhesive and soluble gradients. Adhesive tracks that contain fluorescent strippin and biotinylated RGD were created with micro contact printing or dip pen lithography.
Successful micro contact printing is indicated by the line profile of the fluorescence intensity across the tracks where adhesive tracks and antifouling regions can clearly be distinguished. Dots printed with dip pen lithography appear rounded, not tear shaped, indicating a successful printing process. In this example, dots were printed in a line with row and column separation set to 10 to 15 micrometers.
This distance is sufficient to prevent dots from merging into each other, but allows the viewing of multiple dots in a single field of view. With most microscope settings, a Grady of adhesive cues on the printed track was created with the simple microfluidics device. By loading biotin four fluorescein and biotin RGD into the opposite sides of a microfluidic chamber panel.
A show 60 mosaic images taken with a total image length of 3072 micrometers. In panel B, the fluorescence intensity was measured across the length of the entire mosaic. Linear trend lines were fitted to the fluorescence intensity to indicate the RGD gradient in the channel.
After removing the biotin, biotin RGD solution, the same chamber can be used to introduce a soluble gradient. Panel A shows the fluorescent intensity of fluorescein dye near the PBS and fluorescein filled reservoirs and across the channel at T equals zero. Panel B shows the fluorescent intensity of the soluble cues.
After a 16 hour incubation, the comparable results indicate that the gradient was stable for at least 16 hours. He, a cell migration in a microfluidic chamber with opposing gradients was observed using this method and a representative movie is shown here. The adhesive gradient was immobilized RGD on strep avidan tracks and the soluble gradient was zero to 10%FBS.
The images were taken every 15 minutes for a total of 20 hours. The image transition shown is eight frames per second. This second movie is of a single heli cell migrating towards the FBS source.
The image transition shown is eight frames per second. The blue line representing the cell trajectory was obtained via a cell tracking software. This last graphic shows the cell trajectories from the images in the first movie.
Cell trajectories that migrated towards the higher concentration of soluble FPS are shown in red cell trajectories that migrated towards higher concentrations of adherent RGD are shown in black. These trajectories showed that more hela cells migrated toward the source of the chemo attractant than towards higher concentrations of adherent RGD. While attempting this procedure is important to remember to avoid evaporation of PBS or media in the microfluidic device during the adhesive gradient preparation and lifestyle imaging, use sticky tape if needed to cover all inlets of the microfluidic device Following this procedure.
Other methods like transfection and hybrid solution microscopy can be performed in order to answer additional questions like the biomechanics of the cytoskeleton acting by protein, focal adhesion proteins and receptors during cell migration.
