The overall goal of this technique is to gather mechano biological relevant properties from isolated single cells by visualizing the forces from their characteristic cell substrate interactions using standard light microscopy. The presented method is a platform technology for mechanobiology. It can help investigating key questions in cancer research, such as the role of cell contraction in cancer cell metastasis.
The major advantages of the technique are it robustly extracts neurobiological information with little user training. It is compatible with standard light microscopy and it is geared towards high throughput. Third, this method provides insight into the mechano biological properties of human bone cancer cells.
It can also be applied to auto cell types such as smooth musco cells or cardiomyocytes. Begin by placing the glass substrate so that the micro post array faces up into the well of a 12 Well plate indirectly add one milliliter of 99%ethanol to the well so that the substrate is covered. Make sure that there is no bubble formation during any liquid handling steps, and that the micro post array is always covered by a thin layer of liquid.
Then incubate the array at room temperature for 20 to 30 seconds before adding one milliliter of sterile deionized water on the side of the well. Once it mixes with the ethanol aspirate approximately one milliliter of the ethanol solution from the well and add an additional one milliliter of deionized water. Repeat this process at least three times to wet the sample.
Next, replace the deionized water in the well with PBS in the same manner by adding and then aspirating approximately one milliliter at a time for three repetitions. Then use this same process to replace the PBS with medium. Now that the array is covered with approximately one milliliter of medium pipette 25, 000 cells of interest in one milliliter of medium into the well with a prepared micro post array.
Then close the multi-well plate and transfer it to an incubator at 37 degree Celsius and 5%carbon dioxide. Let the cells adhere and grow on top of micro posts for six to seven hours, inspecting the adhesion process. Occasionally using a light microscope, aspirate the medium from the well and then wash cells twice with one milliliter of PBS.
Make sure to apply gentle force while washing with PBS to remove cell debris that is accumulated on the micro post array during incubation and to detach dead cells. Next, add 0.5 milliliters of a 3.7%buffered formaldehyde solution for five minutes to fix the cells. Then remove the formaldehyde solution and wash the micro post array twice using one milliliter of sterile deionized water.
For each wash, cover the fixed cells with 0.05%kumasi brilliant blue dye in 50%water, 40%ethanol and 10%acetic acid for about 90 seconds, wash away the excess staining solution with two rinses in one milliliter of sterile deionized water. Then add one milliliter of deionized water to the micro post array and check the staining result under a light microscope. Start by adding two milliliters of deionized water to a Petri dish with a thin glass bottom using a pair of tweezers, transfer the micro post array from the 12 well plate into the imaging dish with a micro post facing up.
Then place the imaging Petri dish on the movable stage of a light microscope. If the microscope optics is not infinity corrected, turn the compensation ring of the lens used for imaging until the number on the scale represents the total thickness of all materials along the optical path. Next, remove any face contrast rings from the optical path to enable ordinary bright field mode, and then reduce the iris on the illumination side down to 50%Align the Petri dish so that the micro posts of the array form either a horizontal or a vertical line across the observation field.
Define a start point at the top left and move the Petri dish stepwise across the stage. Scanning the micro post array while taking numerous high resolution images with a 20 x or 40 x objective. Aim to have a single cell in the center of each image at each new position.
Sweep along the ZXs from the micro post bottom towards the micro post tip until the tip is in focus. Using the fine tuning wheel from the microscope. Then lower the focus plane by two to three microns towards the micro post bottom.
Begin by loading the software mec, profiler, and loading in a range of images that need to be analyzed by clicking on open and selecting the images. Next, go to the setting section and enter the parameters as instructed by the software manual. Then analyze the images one by one.
Start by selecting the crop button and then click and drag to form a rectangular outline around the area of interest. When finished, double click inside the drawn rectangle to finish the action. Next, select draw cell outline and use the cross hair cursor to draw an outline around all micro posts covered by the visible cell in the image.
Discard any unwanted micro posts by clicking on to discard posts and use the cross hair cursor for drawing. Then enclose all micro posts that belong to a cell outside of the image section or that are deflected for any other reason, but not by the cell of interest. Next, click on Find OIDs to start the software subroutine.
Adjust the filter setting right next to the find OIDs button until all micro posts are registered, which is made visible by a Red Cross in their center. If there are multiple or missed micro post positions, use the manual editing function by clicking on manual edit and use the mouse cross hair to select the micro post in question. Double click inside the rectangle and place a single red marker in the enlarged image section, which automatically closes.
Find the ideal micro post grid by activating the generate grid function with a mouse click. Ensure the position corresponds with the true micro post head shown by a blue ring inside the drawn cell outline line. Next, correct any misplaced grid markers inside the cell area where needed.
Using the corresponding manual edit function with a mouse click, use the cross hair to select the micro posting question by clicking and dragging it across. Double click inside the appearing rectangle and place the corrected blue marker in the enlarged image section, which automatically closes. Then click on calculate deflections to get a histogram of the calculated deflection values based on the difference between a micro post position inside the image section, and the generated ideal grid.
Finally, save the complete analysis, including the tables of values. Continue the image analysis either by clicking on reset view to analyze another image section or click on next image show.Shown. Here is an example of meno profiling two bone cancer cell lines Q oh nine cells, which have low metastatic potential, and the highly metastatic M 132 cells were seated on a raise from the same production batch.
The outcome shows that Q oh nine cells tend to apply more force at their attachment points than the highly metastatic cells by categorizing the force values. With respect to the cell area, one finds that the average force per cell is elevated for huo nine cells compared to M 132. Also, the average force per micro post increases as the cells spread until the average force per post reaches a more stable value.
Apart from the differences in morphological variances can be quantified with interesting results. For instance, in contrast to the highly metastatic M 132 cells, which tend to occupy fewer micro posts, the HU oh nine cells present a more heterogeneous profile in terms of micro post coverage. In short, the Meno profile for the parental cell line HU oh nine reveals that they typically cover more area and apply slightly more force to the micro posts.
Whereas the metastatic cell line M 132 and aggressive cell type characteristically covers fewer micro posts and applies less force per micro post. While performing this procedure, it's important to remember to pipe it very carefully and never directly onto the micro post array. Avoiding to introduce any bubbles as they lead to the micro posts collapse.
After watching this video, you should have a good understanding of how to extract meno biological information from isolated single cells using micro post Aries in combination with our mesh profile or software. Enjoy your results.
