The overall goal of this procedure is to provide a simple reproducible method for the induction of epithelial to mesenchymal transition or EMT in vitro. This is accomplished by first plating the epithelial cell type of interest in the presence of the EMT inducing media supplement. The second step of the procedure is to replace the culture media with fresh culture media containing the EMT Inducing Media supplement three days after the initial plating.
The final step is to harvest cells five days after the initial plating for downstream characterization, such as immuno staining. Ultimately, results can show cells obtaining mesenchymal characteristics through light microscopy, immuno cyto chemistry, western blood analysis array, or another analysis procedure of choice. The main advantages of this technique over existing methods like TGF beta stimulation or genetic modification, are that this method is quick and easy, unlike genetic modification and allows for EMT induction in a variety of cell types, some of which may not be responsive solely to TGF beta stimulation.
The implications of these technique extend to therapy and diagnosis of cancer metastasis. This technique allows researchers the ability to develop common expression signatures of metastatic cells that could be used in diagnosis. It also provides cells to be used for screening of drugs.
Begin by warming the culture media for the cells of interest to 37 degrees Celsius in a water bath. Next, harvest the cells using a dissociation solution such as with triple E express. Then suspend the cells in Prewarm culture media in a 15 milliliter conical tube, and centrifuge the cell suspension at 400 Gs for five minutes.
Carefully remove the supernatant by pouring it into a waste container and suspend the cell pellet in Prewarm culture media. Now count the viable cells, dilute a sample of the cell suspension in 0.4%Triam blue solution. Place 10 microliters of the diluted sample on a hemo, cytometer and count viable cells.
Viable cells. Do not turn blue after calculating the cell density in your solution plate. Nine to 10, 000 cells per square centimeter in prewarm culture media containing one XEMT inducing media supplement used tissue culture treated plates or flasks culture.
The plated cells at 37 degrees Celsius with 5%carbon dioxide and monitor their morphology daily using an inverted light microscope. Three days later, replace the media with fresh prewarm culture media containing one XEMT inducing media supplement. Five days after plating, the cells are ready for analysis.
Visualize the cells morphology under an inverted light Microscope cells can now be fixed or harvested For downstream studies. Prepare sterile 12 millimeter cover slips for a 24 well plate by placing cover slips in a Petri dish containing 95%ethanol. Gently remove the cover slips from the ethanol using a curved forceps.
Flames sterilize each cover slip and transfer into one well of a 24 well plate handling The cover slips requires care and practice. Next at 0.5 milliliters of Prewarm culture media containing one XEMT inducing media. Supplement to the cells and plate 16, 000 cells per well.
Now grow and feed the cells as previously described. Five days after plating the cells, remove the media and fix them with 300 microliters per well of 4%Paraform aldehyde in one XPBS allow the fixation to proceed for 20 minutes at room temperature. Then remove the fixative and rinse the cells twice with 500 microliters of one XPBS per well.
Next, incubate the fixed cells in 400 microliters of blocking buffer per well for an hour at room temperature. After applying the block, prepare the primary antibody at the manufacturer's recommended concentration in 400 microliters of blocking buffer per well. Then remove the block solution from the wells and add the antibody mix to the cells.
Incubate the cells for either three hours at room temperature or overnight at four degrees Celsius. And if the primary antibody is directly conjugated to a fluorochrome, incubate them in the dark. Next, remove the primary antibody and wash wells three times with 500 microliters per well of one XPBS containing 0.1%BSA allow each wash to go for five minutes and if needed, shield the cells from light.
Now if needed, incubate cells in a secondary antibody at the manufacturer's recommended concentration. Dilute the antibody in 400 microliters per well of one XPBS containing 1%BSA. Allow a one hour incubation at room temperature shielded from light after an hour.
Wash the cells three times just as was done to remove the primary antibody after the washes. If desired, apply a DPI counter stain to the cells. Lastly, rinse the cells in deionized water and mount the cover slips face down on slides using mounting media.
Again, be careful in handling the cover slips. The EMT inducing culture conditions provide a robust method for the induction of EMT in a variety of cell types. This was tested on four different human cell lines.
Cells that were treated with the EMT inducing media supplement changed from a classical epithelial morphology to a mesenchymal spindle shaped morphology. Double staining for e cadherin and fibronectin expression demonstrated the downregulation of the epithelial marker e cadherin in red and the upregulation of the mesenchymal marker fibronectin in green un induced CF 10 A samples contained tightly packed clusters surrounded by more loosely packed cells. These clusters were E could herein positive shown in red.
The clusters disappeared upon treatment with the EMT inducing media supplement. This coincided with an increase in fibronectin expression in green. T 98.
G was found to have an extremely low basal level of terrin prior to EMT induction, which precluded its analysis. With this marker, however, fibronectin levels were found to increase significantly with EMT induction in these cells. The expression levels of e adhere and fibronectin were further confirmed through western blotting of total cell lysates.
Although the western blot does not show significant reduction of total EAD herrin protein levels in HT 29 cells, there was a reduction in surface expression of terrin seen by Immunochemistry to further evaluate EMT status mesenchymal markers characteristic of EMT menton in green and snail in red were analyzed pre and post EMT induction consistent with previous results. Eki herein in gray was downregulated in all cell lines examined indicating that EMT was induced. In addition, there was upregulation of snail in red and menton in green in a 5 49 T 98 G and MCF 10 A cells CCF seven human breast cancer cells and PanIN human pancreatic carcinoma cells are both reported not to enter EMT by TGF beta signaling alone.
These reports were confirmed with recombinant TGF beta one alone. At the concentration within the EMT inducing media supplement, the cells retained their epithelial morphology and surface E coherent levels were similar to the control cells. In contrast cells stimulated with the EMT inducing media supplement showed a drastic decrease in their surface E cadherin levels.
These cells also obtained a more mesenchymal morphology. Another hallmark of mesenchymal cells is their ability to migrate and invade. This was analyzed using the 96 well BME cell invasion assay.
According to the manufacturer's instructions, significant increases in cell migration were seen with both a 5 49 and pan one cells. Following EMT induction, the same assay was performed with a basement membrane extract coated filter to test invasion. EMT induced cells showed a significant increase in invasion capabilities compared to untreated cells.
The robust induction of EMT is useful for the analysis of gene expression changes and signaling taking place in these cells. A commercial antibody-based array using lysates from MCF seven and a 5 49 cells was used to analyze the levels of phosphorylated MA kinase family members. Both cell types exhibited increased phosphorylation of kreb irk one and irk two in EMT induced cells compared to the controls.
A 5 49 cells also showed an increase in GSK three beta phosphorylation. Mc seven cells showed increased P 70 S six K phosphorylation in addition to the increased phosphorylation of kreb or one and DIRK two. While attempting this procedure, it's important to remember that there can be some variability in results from different cell types, not all cells.
We use the same downstream pathways for EMT induction leading to differences in marker expression between cell types. Additionally, many markers of EMT do not demonstrate strict on and off states and may have basal levels in epithelial cells. Such markers can show an increase interchange of localization upon mesenchymal transition Following this procedure.
Other methods like drug screening can be used to answer additional questions like what compounds are able to modulate epithelial to mesenchymal transition. These compounds could be very useful in the treatment of cancer and fibrosis.
