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.