The overall goal of this procedure is to be able to quantitatively measure cellular motility over time using a gold nanoparticle based cell motility assay. This is accomplished by first preparing glass cover slips for a coating of gold nanoparticles. Specifically endotoxin free cover slips are coated with gelatin and then baked.
The second step is to reduce chloro oric acid, which will create gold nanoparticles and to coat the gelatin coated cover slips evenly with the solution. Next, the cells are placed onto the gold nanoparticle coating cover slips for the desired timeframe of the experiment cells. Moving on, the gelatin will displace gold nanoparticles creating tracks.
The final step is the monitoring and quantitation of cell motility using microscopy to image the tracks created by the moving cells. Ultimately, area measurements of the tracks are performed using freely available software. One of the main advantages of this technique over existing methods, such as time lapse imaging, is that this assay requires only a standard light microscope, standard camera, and uses freely available imaging software.
In addition, the assay also easily allows the analysis and comparison of multiple samples using a Methodist capable of high throughput screening. Although this method can provide insight into the influence of different physiological factors and cell movement, it can also be applied to other systems such as the studies on the effects pathogens have on the motility of infected cells. Generally, individuals new to this method may struggle because the methods requires one to strictly follow volumes and temperatures indicated in the protocol.
In this protocol, an emphasis is placed on assessing the color of the final solution of precipitated gold nanoparticles to ensure success. Visual demonstration of this method is critical. As the FGO kinetic track motility assay steps are difficult to learn.
They contain several procedures not commonly used in cellular and molecular biology laboratories. To prepare gelatin coated cover slips, first Resus suspend the gelatin and deionized water to make the solution with a final concentration of 0.5 grams per 300 milliliters then autoclave the mixture for 15 to 30 minutes. It is important to do this within two hours of the addition of the gelatin in a tissue culture hood.
Prepare the cover slips for coating by first transferring eight to nine acid washed cover slips into a 100 millimeter plastic dish. Make sure the cover slips are not touching each other or the sides of the dish. Carefully pipette two to three drops of gelatin onto each cover slip avoiding overflow onto the plate.
Then place the dish containing the gelatin coated cover slips into an oven and bake at 90 degrees Celsius for 10 minutes. After 10 minutes, remove the dish from the oven and return it to the tissue. Culture hood.
Gently pipette off any excess gelatin. Return the dish to the oven and dry the cover slips at 70 degrees Celsius for 45 minutes. Once the cover slips are dry, remove the dish from the oven and return it to the tissue Culture hood.
Using a sterile needle delicately, lift up one side of the cover slip. Then use sterile tweezers to remove the gelatin coated cover slips from the dish and place them into separate wells of a 24 well plate. The next step is to prepare colloidal gold coated cover slips.
Begin by preparing 10 milliliters of 0.5%sodium citrate solution working in a tissue culture hood combined 1.5 milliliters of the 14.5 millimolar chloro ORIC acid solution, and 13.5 milliliters of autoclave deionized water in a sterile endotoxin free erlenmeyer flask For every eight to nine cover slips, the result should yield a faint yellow solution. Chloro ORIC acid is toxic and should be handled carefully. It is also light sensitive, so the boiling step should be carried out in a low light setting.
When the solution has reached its boiling point, remove the flask from the hot plate and transfer it to a stir plate. In order to form the colloidal gold particles in solution, add 0.7 milliliters of the 0.5%sodium citrate solution per 15 milliliters of the chloro ORIC acid solution while stirring, continue stirring for approximately two minutes. During this time, the solution will change from a faint yellow to clear to gray to purple to deep purple.
Before finally reaching a red wine or preferably rust color, draw up the colloidal gold solution in a 10 milliliter pipette and allow the solution to cool in the pipette for one to two minutes until it reaches room temperature. Then add 0.5 to one milliliter on top of the gelatin coated cover slips in the 24 well plate Place the 24 well plate containing the colloidal gold covered cover slips in an incubator at 37 degrees Celsius and incubate for 30 minutes. After the particles have had time to settle onto the gelatin coated cover slips, check their density under a light microscope.
The appropriate distribution of gold nanoparticles helps to easily and efficiently distinguish the edges of solar trucks. The optimal density varies with cell size. A concentration of gold particles that is too high hampers the ability of cells to move while a concentration of gold, gold particles that is too low limits the ability to delineate an accurate track of motility.
For this reason, only cover slips made in the same batch or that are ized to have the same density should be used together in individual experiments. If the concentration of gold particles is insufficient, add an additional 0.5 to one milliliter of the colloidal gold solution to the gelatin coated cover slips. If however, the concentration of gold particles is too high, such as the case shown here, the only effective choice is to remake the colloidal gold solution and coat new cover slips.
If the particle density is correct, return the plate to the incubator at 37 degrees Celsius and incubate for one hour to overnight rinse away unbound gold particles by dipping the cover slips three times into sterile phosphate buffered saline. Then store the colloidal gold coated cover slips in PBS filled wells of a clean 12 well plate at four degrees Celsius until they're ready to be used. Cover slips should not be allowed to dry and should be used within two to three months from the date they were made.
The next step is to prepare the colloidal gold coated cover slips for cell culture. Begin by placing them into individual wells of a 24 well plate that have had approximately 0.3 milliliters of culture media added prior to the addition of the cover Slips transfer approximately 5, 000 to 50, 000 cells onto each colloidal gold coated cover slip. The cell number will vary by cell type, but the cell density must be low enough to prevent the overlapping of cell tracks from multiple cells in the same area.
Cover the plate and place it in an incubator at 37 degrees Celsius with 5%carbon dioxide for six to 24 hours Based on the motility of the cell type, the optimal timeframe should be experimentally determined for each cell type following incubation, fix any cover slips that will not be analyzed immediately by first dipping them twice in PBS followed by incubation in 3%para formaldehyde for 15 minutes at room temperature using a light capture images of the tracks created by a single moving cell on unfixed or fixed cover slips. The magnification used to take pictures of cellular tracks varies depending on the cell type. However, the same magnification must be used for each study in order to be able to compare results.
For monocytes, 40 x magnification works well using the freely available software such as image, JNIH image or image tool determine the average area of colloidal gold cleared by 10 to 20 or more cells for each sample from the captured images shown here is the cleared path of colloidal gold particles by a single unstimulated monocyte taken with a 40 x objective. Non motile cells create characteristic small oval or circle shaped tracks around themselves, which indicates a low vassal level of movement. In contrast, highly motile cells are characterized by a directional movement as elongated tracts and larger overall areas due to their irregular shapes.
Cell tracks are best analyzed by using the freehand tool in the Image J software to outline their volume. Quantitative area measurements can then be performed using the measure tool in the analysis menu of Image J.While attempting this procedure, it's important to remember to strictly follow the volumes and temperatures indicated in the protocol as deviations can strongly influence the final results. After watching this video, you should have a good understanding of how to create gelatin coated cover slips, how to prepare gold nanoparticles and gold nanoparticles coated cover slips, and how to monitor and quantitatively evaluate cell movement using a light microscopy.
Don't forget that working with the boiling solutions on a hot plate, especially vapors from the boiling solution of fluoro, AIC acid and sodium citrate can be hazardous precautions such as working in the hood and using common sense should always be taken while performing this procedure.
