To begin, seed split our loop transfected HEK293 T cells in a Poly-D-Lysine coated 96 well plate. Next, prepare three fresh solutions of Rhosin, Ehop-016, and ZCL278 in a 1.7 milliliter Microfuge II.Add live cell substrate for renilla luciferase, diluted 1 to 2, 000, to a final concentration of 30 millimolar to each solution. To make 0.55 and 50 micromolar ZCL278 solutions, perform serial dilutions using the initial 50 micromolar ZCL278 in culture media.
Then remove the media from each well of the 96 well plate containing transfected cells. Add 50 microliters of the chemical and substrate media mixture to each well. After incubating the cells for one hour, two hours or five hours, load the plate onto the luminescence plate reader and measure luminescence.
Ensure the plate reader is turned on before starting. Access the Microplate Reader Software and click new session to create a new file. Click incubator to set the plate reader's temperature to 37 degrees Celsius.
Check the temperature option and type 37 degrees Celsius. Under plate layout, choose the option unknown and click and drag the well to specify the wells to measure. Then go to protocol, click luminescence, and keep the default settings.
Once the setup is complete, load the plate with the lid off into the plate reader and choose run plate in. Click start and a save file window will appear. Rename the file and click save.
Once the reading is complete, remove the plate from the plate reader and then click the run plate in option to put the reader back in. Once completed, place the plate back in the humidified incubator until the next reading. At one, two, and five hours of the treatment, Rhosin treated cells showed no significant changes in luciferase activity compared to control DMSO treated cells.
The Rac GTPase Inhibitor EHop-016 showed significantly lower luciferase activity than DMSO. ZCL278 treated cells had significantly lower luciferase activity than the control DMSO at all three time points. Luciferase activities measured at one, two, and five hours post-treatment showed a dose-dependent response.
Higher concentrations of ZCL278 still showed significant changes compared to the control DMSO.
