Differential Nuclear Staining Assay: An Assay to Determine Mitocan Cytotoxicity by Propidium Iodide and Hoechst Double Staining

Begin by preparing solutions of the compounds of interest at the desired concentration. Always make sure to mix compounds by vortexing thoroughly.

To measure cytotoxicity of a single compound, prepare compounds at 2X final concentration. If measuring cytotoxicity of compound combinations, prepare them at a 4X final concentration.

Prepare solvent-only controls by mixing the same amount of solvent with the appropriate medium. Collect cells into a 15-milliliter conical tube, and aliquot 10 microliters of the cell suspension for trypan blue staining. Add 10 microliters of 0.4% trypan blue to the aliquot, and use a hemocytometer or cell counter to count viable and non-viable cells.

Pellet cells in the 15-milliliter tube at 200 x g for 5 minutes, then, aspirate or decant the supernatant. Resuspend the cell pellet in assay-appropriate media at a cell density of 3x10⁵ cells per milliliter. Use a multi-channel pipette to seed 50 microliters of cell suspension into each well of a 96-well plate.

For single compound assays, add 50 microliters of 2X compound solution into each well. For solvent-control wells, add 50 microliters of test media containing the solvent at 2X concentration. For combination assays, add 25 microliters of each of the 4X compounds into each well. For single compound-control wells, add 25 microliters each of 4X compound solution and test medium. Make sure that the final concentration of DMSO does not exceed 0.5%.

To ensure the reproducibility, add drugs with the pipette tip touching the side of wells, being careful to add the drugs to different locations. This step should be performed very quickly, preferably taking no longer than 30 minutes per plate.

Gently tap the plate to mix the contents of the wells, and incubate at 37 degrees Celsius and 5% carbon dioxide.

When ready to stain the cells with Hoechst 33342 and propidium iodide, prepare fresh 10X staining solution, and add 10 microliters to each well. Gently tap the plate, and incubate it at 37 degrees Celsius for 15 minutes.

Centrifuge the plate at 200 x g for 4 minutes to bring all the cells to the bottom of the plate. Then, carefully wipe the bottom of the plate with a damp laboratory wipe to remove any debris that may interfere with imaging. Image the plate within 15 minutes after centrifugation.