The function of ion channels is crucial in cell biology and significantly impacts cancer growth. Our team is focused on targeting ion channels to aid cancer treatments. The field has recently gained a greater understanding of the tumor microenvironment and how cancer cells take hold in healthy tissue, highlighting the importance of ion channels in cancer.
One of the biggest challenges in current experiment is the availability of immune-competent animal model that can effectively test therapeutic treatments. We have established that GABAA receptor is a therapeutic vulnerability to disparate cancers and identified a new class of GABAA receptor activators that effectively impair cancer cells. Ion channels play a crucial role in regulating cancer cell behavior, and they hold great potential as target for cancer treatment.
Our research has highlighted how an established class of drug can be employed to treat cancer effectively. To begin, maintain cells in a 75-square centimeter culture flask. Aspirate the culture medium and rinse the cells with PBS without calcium and magnesium.
Next, add two milliliters of 0.25%Trypsin-EDTA to detach adherence cells. Incubate for five minutes at room temperature and re-suspend the cells in 10 milliliters of medium. Next, collect the cells from the flask into a 15 milliliter centrifuge tube, centrifuge at 480G for five minutes at room temperature, and aspirate the medium.
Re-suspend the cells in five to 10 milliliters of medium based on the original confluency of the culture. Remove 100 microliters of cells and add to a 1.5 milliliter tube with 100 microliters of 0.4%Trypan Blue. Add the cells to the hemocytometer and count them by observing under the microscope and using the manual tally counter.
Dilute the cells to three times 10 of the fourth cells per milliliter in the culture medium without phenol red. Add 2.5 milliliters of the cell suspension to each well of a six well plate with Poly-D-Lysine coated cover slip per the manufacturer's directions. Grow the cells overnight at 37 degrees Celsius and 5%carbon dioxide in a humidified incubator to enable cell attachment to the cover slip.
The next day, examine cell attachment under an inverted microscope using a 20 times objective. Prepare the treatment stock solutions at the desired concentrations and create a media only control, a vehicle only control at a concentration that matches the test compound, and a control with the ionophore FCCP and the test compounds. Working with one cover slip at a time, add 1.8 milliliters of medium for the condition under the study to the cells.
Incubate the cells treated with either the control or test compound at 37 degrees Celsius and 5%carbon dioxide in a humidified environment for the desired time. Following the treatment period, add 200 microliters of 10x TMRE to the cells. Incubate for 15 to 30 minutes at 37 degrees Celsius in a 5%carbon dioxide humidified environment.
Gently aspirate the medium and rinse the cells two times with PBS to minimize background interference. Then invert the cover slip onto a microscope slide labeled with the treatment conditions. Image the cells live at 549 nanometers excitation and 575 nanometers emission.
Utilize a confocal microscope to capture several Z-stack fields with high speed image capture before the cell integrity is lost. Save and store the image file for later analysis. The cells with active mitochondria retained TMRE staining and showed high fluorescence.
Depolarized or inactive mitochondria have reduced membrane potential, failed to retain TMRE, and show a low fluorescence signal. To begin, plate the cells in 75 microliters per well of antibiotic-free medium without HEPES buffer to determine the IC 50. On the second day, prepare the control solution and the test solutions by performing serial dilutions of high concentration stock solutions.
After adding control and test solutions to the cells, incubate the cells at 37 degrees Celsius and 5%carbon dioxide in humidified environment for 48 hours. Begin the cell proliferation assay by first vortexing the MTS reagent to fully dissolve any precipitated reagent before usage on day three of the experiment. Transfer the thawed MTS reagents into a sterile 25 milliliter reagent reservoir.
Pipette 20 microliters of MTS reagent into each well of the 96 well plate containing samples and 100 microliters of culture medium. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide in a humidified environment for one to four hours. Then centrifuge the plate at room temperature to remove any bubbles that may interfere with the absorbance reading.
Measure the absorbance at 490 nanometers with a microplate reader or spectrophotometer. A 96 well plate showing the colorimetric results of an MTS assay with increasing drug concentrations is presented. The dose response curve showed that the test agent impairs the viability of the cancer cells in a dose dependent manner.
