The overall goal is to provide a simple assay to measure gap junctional intercellular communication through a population of mammalian cells that can serve a broad range of in vitro pharmacological and toxicological needs. This method can help answer key questions in the toxicology field, such as whether a compound affects the development or maintenance of a tissue, by disregulating gap junctional intercellular communication. The main advantage of this technique is that it offers a simple and quick assessment of gap junction function in a large population of cells in response to toxicants and toxins.
Demonstrating the procedure will be Ajna Uzuni, an undergraduate research assistant from my laboratory. To begin the experiment, seed three times 10 to the fifth WB-F344 rat liver epithelial cells onto 35 millimeter diameter culture plates containing Eagle's modified medium plus 5%fetal-bovine serum. Culture the cells in an incubator until they reach 100%confluence and continue with the experimental treatment of the cells.
To continue with dose-response experiments, add 10 microliters and 20 microliters of a previously prepared, two millimolar stock solution of phenanthrene in 100%acetonitrile, and four microliters, six microliters, and eight microliters of a 20 millimolar stock solution of phenanthrene in 100%acetonitrile to three plates of cells containing two milliliters of growth medium for each added volume of stock solution. Next, add four microliters, six microliters, eight microliters, 10 microliters, and 20 microliters of a 100%solution of acetonitrile to three plates of cells containing two milliliters of growth medium for each added volume of acetonitrile solution to serve as the vehicle control. After adding the acetonitrile, incubate the plates.
To perform time response experiments, add seven microliters of a 20 millimolar stock solution of phenanthrene in 100%acetonitrile to three plates of cells, containing two milliliters of growth medium for each time point. Then, add seven microliters of 100%acetonitrile to three plates of cells, containing two milliliters of growth medium for each time point, to serve as the vehicle control. Incubate the plates for each designated time point.
Discard the culture medium by gently pouring off the medium. Rinse the cells with three milliliters of PBS, and either decant or aspirate between rinses. Pipette one milliliter of one milligram per milliliter lucifer yellow dye dissolved in PBS into each cell plate.
Preload the dye into the cells by pinching the scalpel between the index finger and thumb. Place the scalpel perpendicular to and five millimeters from the edge of the culture plate and then use gravity to allow the scalpel to gently roll from this perpendicular angle to an angle of 15 degrees. Roll the blade through a population of cells in three different areas of the total plate.
Next, incubate the cells with the LY solution for three minutes at room temperature. Decant or aspirate the LY and rinse the cells to remove all extracellular dye and eliminate extra cellular background fluorescence. Then, add 0.5 milliliters of a 4%phosphate buffered formalin solution to fix the cells.
View the fixed cells using an epifluorescence microscope at 200x magnification. Align all of the plates so that the indentation line is parallel to the horizontal field of vision. A series of SL-DT images were generated of WB-F344 rat liver epithelial cells treated with increasing doses of phenanthrene.
The decrease in lucifer yellow fluorescent dye migration is caused by increasing doses of phenanthrene, demonstrating the increasing toxicant-induced inhibition of gap junctional intercellular communication, or GJIC. The fraction of the control, or FOC values, obtained from the SL-DT images were averaged, statistically analysed, and then graphed. Following this procedure, other methods like western blots of single transduction proteins can be performed in order to answer additional questions.
Like, are these compounds activating mitogenic events of cell perforation?
