The overall goal of the following experiment is to establish an in vitro assay for examining the role of hormones, cytokines, and or growth factors in driving cancer cell invasion. This is achieved by determining whether there is a difference between the invasion index of different cell variants or mutants as a second step. The invasion assay is repeated using charcoal stripped media, which removes any hormones, growth factors, or cytokines from the serum.
The invasion assay is then repeated with the addition of a specific known factor to the charcoal stripped medium to determine whether migration toward that factor enhances or inhibits invasion. The results show the changes in the invasion index for specific cell populations that occur during migration toward known experimental factors based on the normalization of the results against the invasive index of non-invasive cell populations. I first got the idea for this method when I was performing a traditional invasion assay, and I noticed that there was a significant difference between the invasion indexes for different cell mutants.
The main advantage of this technique over existing methods like the traditional invasion assay, is that with this technique can specifically investigate the role of hormones, specific growth factors and cytokines in promoting or inhibiting cell invasion. This method may be helpful in answering key questions in the field of cancer research, including which factors lead to cancer metastasis. The Implications of this technique may provide a therapeutic approach to future drug design For each cell line to be tested, use a 24 well plate with 12 inserts.
Then label the plates according to the conditions to be analyzed. Next carefully, dispense 75 microliters a freshly prepared collagen matrix solution into each insert that will be used for an invasion assay taking care not to make bubbles. Then incubate the plates with the collagen coated inserts at 37 degrees Celsius and 5%carbon dioxide for 30 minutes to allow the gel to solidify.
After detaching the experimental cells with trypsin, add medium supplemented with 10%FBS to inactivate the enzyme and wash the cells three times in PBS. Then resuspend the pellet in serum free medium and count the cells. Add serum free media to a final concentration of 50, 000 cells per milliliter when the collagen matrix has solidified.
After 30 minutes, add 700 microliters of medium with either 2%defined FBS 2%charcoal stripped FBS or 2%charcoal stripped FBS plus the additional component into each. Well add 500 microliters of cell suspension in serum free medium into the top of each migration or invasion. Insert, incubate the assays for 22 hours at 37 degrees Celsius.
Then add methanol, fixative, eoin, and hematin into three of the empty wells of the chamber plate. Next, remove the cells and matrix from the top of each insert with two sequential cotton swabbing. Then using forceps.
Dip each insert five times for one second each time into each of the three staining solutions in succession, and allow the inserts to dry overnight inverted. The next day. Use a 20 x objective to acquire images of the cells on the inserts by directly viewing the stained inserts.
Five images are taken for each sample including four from the outer fields of the membrane and one from the center. Finally, using image J software, count the cells for each image and calculate the invasion index as normalized to a non-invasive cell line. This model demonstrates the potential results that may be encountered during an invasion assay and how to interpret the corresponding data.
Each circle illustrates a representative result for a stained filter from one of the three conditions tested, and each dot represents a cell that passed through the invasion matrix and was stained on the bottom of the membrane. The invasion index is then calculated for each condition according to its normalization to a non-invasive cell line. For example, in this representative experiment, three hormones were tested to identify a possible x factor.
Two of the hormones had no effect on the invasion index, but one was identified to exhibit inhibitory properties Once mastered. This technique can be done in approximately four hours on day one and one hour on day two if all steps are performed properly. While using this technique, it's important to properly label each individual cell insert carefully.
We used a color coded system and different dots to carefully label each cell insert Following this. Other methods like in vivo, mouse models for metastasis could be used to validate the in vitro results. After watching this video, you should have a good understanding of how to assay the physiological relevance of factors such as hormones, growth factors, and cytokines as either enhancers or inhibitors of cell invasion in an in vitro model.
Thank you for your interest in our video and good luck with your experiments.
