The overall goal of the following experiment is to use a measure the effect of potential regulators of TGF beta induced invasion in a three-dimensional environment. This is achieved by first making spheroids of the cells. The PHE are then embedded in collagen, which provides a 3D environment.
Next, the invasive area is quantified. In order to determine the effect of treatment on TGF beta induced invasion, the results show that modulators of the TGF-beta pathway affect the invasive property of the steroids in a 3D collagen matrix. The main advantage of this technique over existing methods like the scratch ass essay, is that it better recapitulates the 3D environment.
This method can help to answer key questions in the T meter fields, such as the identification of novel genes involved in teacher beta induced invasion. To prepare methyl cell solution for the TGF beta induced invasion assay begin by autoclaving six grams of Methylcellulose in a 500 milliliter bottle containing a magnetic stir. Dissolve the Methylcellulose in 250 milliliters of preheated D-M-E-M-F 12 without serum for 20 minutes, add 250 milliliters of room temperature D-M-E-M-F 12 containing 10%horse serum mix overnight at four degrees Celsius the next day, clear the solution by centrifugation at 5, 000 G for two hours at four degrees Celsius.
Transfer the clear, highly viscous solution to a new tube store at four degrees Celsius until use to culture phe. Prepare a 20%methyl cell solution using 10 milliliters of the prepared methyl cell stock and 40 milliliters of growth. Medium wash MCF 10 A cells twice with PBS.
Add 0.1%trypsin and incubate the cells at 37 degrees Celsius until all cells are detached. Resuspend the cells in 10 milliliters, growth medium, and count the cells using a cell counter. Prepare a suspension of 10, 000 cells per milliliter in 20%methyl cell.
For each test condition, add 100 microliters of the cell suspension to 12 wells of a 96 well round bottom suspension plate. Incubate the cells overnight in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide. The next day check for spheroid formation.
Spheroids will be ready for use within one to three days. Prepare on ice a solution of eight milliliters of collagen, one milliliter of 10 XPBS, one milliliter of 0.1 normal sodium hydroxide, and three drops of 0.1 normal hydrochloric acid. Check that the solution's pH is 7.4 by spotting it on pH indicator strips.
Adjust the pH if it is out of range. Add 50 microliters of neutralized collagen solution to the wells of a 96 Well plate incubate at 37 degrees Celsius for about 90 minutes or until the collagen is solidified. For each ligand to be tested, prepare a methyl cell collagen ligand solution on ice.
Begin with one milliliter of collagen solution per treatment, and add 20 nanograms of TGF beta for a final concentration of five nanograms per milliliter mixed by vortexing. Then add one milliliter of methyl cell solution for a final volume of two milliliters and vortex. Again to embed a steroid place a 200 microliter pipette tip, which had five millimeters or more of the tip cut off on a 200 microliter pipette.
Then take up one s spheroid with the pipette. Carefully dispense the medium into an empty plate while ensuring that the S spheroid stays inside the tip. Do not release the plunger.
Take up 100 microliters of the collagen methyl cell mixture and dispense the S spheroid collagen mixture into a collagen coated well of a 96. Well plate fill 12 wells for each condition. Let the collagen solidify at least 30 minutes at 37 degrees Celsius and remove the air bubbles with a 200 microliter tip.
Prepare a 40 micromolar solution of the TGF beta inhibitor SB 4 31 542 by adding four microliters of SB 4 31 542 stock solution to one milliliter of D-M-E-M-F 12 containing 1.6%horse serum. Add 50 microliters of medium with inhibitor to each well after diffusion. The final concentration of the inhibitor will be 10 micromolar.
Take pictures of the steroid under the microscope using 40 x magnification, then incubate in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide. After a two day incubation, take pictures again and measure the area of the invading steroid by opening a spheroid picture in Adobe Photoshop. Extended and select the quick selection tool.
The mouse pointer will change into a circle with a plus sign while dragging the cursor over the spheroid. The software will recognize the borders of the spheroid. If an area outside the steroid is selected, exclude the area by pressing the alt key or by using the negative selection tool, which is a negative sign in the toolbar.
Drag the cursor over the wrongly selected area and the software will remove it from the selection. If an area within the spheroid is deselected, the area can be included again by dragging while pressing the shift bar. To work more accurately, adjust the size of the mouse pointer by changing the brush diameter in the toolbar.
To calculate the area of all active selections, choose analysis measure In the menu bar. A measurement recording window will appear showing the file name and the area of selection in pixels. If multiple selections are measured within one spheroid, the first line will show the sum of all the areas and the lines below.
It will list the values of the individual selections. Export the data into a text file and open it in excel. An example of the S spheroid assay with MCF 10 A one, normal breast epithelial cells, HAS transformed MCF 10 A neo T cells and M two derived MCF 10 CA one A is shown here.
M1 cells showed only weak invasion without stimulation, but invaded significantly better after stimulation by TGF beta. In contrast, the RAs transformed M1 derivative M two invaded efficiently without the addition of stimuli, and this was further increased four to fivefold. After TGF beta treatment, M four cells showed the strongest invasion both without and with TGF Beta Edition.
A graphical representation of the results for this experiment was obtained using Photoshop and is shown here in this example, SB 4 31 5 42 an A TP analog and selective inhibitor of the kinase activity of TGF beta receptor one, as well as active in type one B receptor and active in receptor like kinase seven was added to the cell cultures SB 4 31 5 42, potently inhibited, TGF beta induced invasion of M1, M two, and M four cells, indicating that TGF-beta induced invasion is TGF-beta receptor one kinase dependent. In addition, the basal invasion of the PHE was strongly inhibited. A graphical representation of the results for this experiment was again created using Photoshop and is shown here.
Following this procedure, other methods like RNA isolation or protein isolation can be performed to answer additional questions like which genes are expressed or which proteins are expressed. After watching this video, you should have a good understanding of how to find new modulators of tavita induced invasion.
