In 24-well plate- seed 25,000 cells/well in 1 mL media (SUM1315- F12+I/E)
Next day add compounds
(Dilute the ~12mM stock solution as follows: Take 1µl of the stock and add it to 999µl of DMSO. This will give you a 12µM stock solution. Next take 50µl of this stock solution and add it to 550µl of DMSO. This will give you a 1µM solution, This is the stock solution you should add to the cells. For each well, add 1µl of this stock solution.
Use 1 µl of DMSO as a control. (See picture for plate layout)
At time points listed below do the following:
1) Collect media in well and put into a conical tube.
2) Trypsinize the cells and collect these cells in the same tube as 1). Add 50µl of Typan blue solution to the tube and count the cells using a hemocytometer. Make sure to document the number of blue cells and the number of non-blue cells.
Count cells at 1hr, 3hr, 24hr, 48hr and 72hr.