This experimental system aims to characterize the contribution of specific proteins to the invasive process. Begin with retroviral transduction of cells with the fluorescent protein of interest. Also create the 3D matrix of matrigel protein in transwell inserts, and prepare a cell suspension at the appropriate dilution.
Using the Matrigel transwell system, measure the transmigration response to the chemo attractant with test drugs or treatments proceed to visualize and analyze cell invasion by confocal microscopy. Ultimately, results can determine the magnitude and mode of cell invasion into three dimensional protein matrix. The power of this technique over existing methods like the traditional Transwell invasion assay, is its capability to drive both quantitative and qualitative data on cell invasion into a three-dimensional protein matrix.
My collaborator, Diane Creighton, will now demonstrate the method Culture retroviral packaging cells in DMEM containing 10%FBS for 48 hours. Transfect the cells with retroviral DNA according to the manufacturer's instructions. Incubate for 24 hours, rinse the wells twice with medium, then add 1.5 milliliters of 10%F-P-S-D-M-E-M per well, and incubate for 48 hours to collect the package Retrovirus.
Transfer the tissue culture medium to two milliliter micro centrifuge tubes, centrifuge to pellet out any cells and harvest the supinate into a clean tube. Store this retrovirus stock at minus 80 degrees Celsius. Now to retro transduced cells, remove media from an overnight culture and add one milliliter of retrovirus stock supplemented with poly green.
After a six hour incubation, add two milliliters of 10%F-B-S-D-M-E-M to the well and place the plates in a 37 degrees Celsius incubator. Overnight, replenish the media and after 24 hours, add appropriate selective media when confluent harvest the cells and sort for fluorescence using nont transduced cells as a reference. Collect cells with fluorescence greater than nont transduced cells and freeze aliquots at minus 80 degrees Celsius for future use.
This method relies on expression of fluorescent proteins for visualization and allows for the insights into the contributions of individual cells to be determined. Thor complete matri gel on ice and then dilute one to one in ice cold PBS. Now insert the uncoated transwells into a 24 well tissue culture plate.
Then carefully pipette 100 microliters of diluted matri gel into the wells and solidify at 37 degrees Celsius. Meanwhile, prepare the fluorescently labeled cell suspensions in their normal growth medium. When the matri gel has solidified, invert the transwells and pipette 100 microliters of the cell suspension onto the output facing underside of the filter.
Carefully cover the transwells with the base of a 24 well tissue culture plate making contact with each droplet of cell suspension. Incubate the plate in the inverted state for four hours to allow for cell attachment. Now turn the plates right side up and wash each transwell by dipping into one milliliter of serum free medium twice.
Then position the transwell in a third well containing any drugs or treatment reagents. Agents gently pipette 100 microliters of media containing chemo attractant into the transwell on top of the solidified matrigel PBS mixture. Incubate for three to five days at 37 degrees Celsius with 5%carbon dioxide to image non fluorescent cells invading Matrigel.
Place transwells in fresh 24 well dishes and pipette. One milliliter of four micromolar calcium AM solution on top of each matri gel plug, allowing it to spill over the sides and stain from the top and bottom Incubate for an hour at 37 degrees Celsius in 5%carbon dioxide humidified atmosphere. Then image by confocal microscopy.
This first lifestyle staining method allows for a visualization of invasion modes. Transfer each transwell to a fresh 24 well plate overlay, one milliliter of 4%paraform aldehyde 0.2%triton X 100, and incubate at room temperature for half an hour. After washing three times with one milliliter, PBS add RNAs A for 30 minutes, then wash twice with PBS for visualization at 0.01 milligram per milliliter.
Propidium iodide diluted in PBS and leave it room temperature in the dark for 30 minutes. Wash three times with PBS. At this stage, the propidium iodide stain transwells can be kept at room temperature in the dark for at least one month.
The second cell staining method requires fixation, but allows for more rapid quantification of invasion Using an inverted confocal microscope placed transwell with a small amount of residual PBS onto a large cover slip over non immersion 20 times objective and capture optical sections every 10 to 15 microns from the bottom of the matrigel plug. Quantify the extent of invasion using individual optical slices. Also construct three dimensional objects using appropriate software such as velocity in this said series of optical slices, cells was stained with calcium am.
As expected, the number of cells invading up from the filter decreases with distance. This invasion can be quantified. Three dimensional reconstructions of cell invasion offer a visual depiction of the mode of invasion.
If cells are labeled by expression of fluorescent proteins, then the positions of each color cell can be visualized in three dimensional reconstructions as shown in this side view. In addition, slices through the reconstruction can offer insights into molecular mechanisms. After watching this video, you should have a good understanding of how to use this cell invasion assay to obtain more information about the evasive process than can be obtained with the standard methods.
