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

1. Cytotoxicity Assay: Setup

1. Prepare solutions of compounds of interest at desired concentrations in the appropriate media (serum-free or 1, 2.5, or 5% FBS RPMI-1640).
   1. To measure the cytotoxicity of a single compound (e.g., to determine effective doses), prepare compounds at 2x final concentration.
   2. To measure the cytotoxicity of compound combinations, prepare compounds at 4x final concentration.
   3. Prepare solvent-only controls by mixing the same amount of solvent with the appropriate medium. For example, if testing compounds dissolved in DMSO and methanol, make solvent-only control for each solvent.
2. Collect cells from the culture dish or flask into a 15 mL conical tube.
3. Transfer 10 µL of cell suspension into a microcentrifuge tube and stain with 10 µL 0.4% trypan blue. Use a hemocytometer to count viable and non-viable cells for each cell source.
4. Pellet cells at 200 g for 5 min. Aspirate or decant supernatant.
5. Resuspend cell pellet in assay-appropriate media (serum-free or 1, 2.5, 5% FBS RPMI-1640) at a cell density of 3*10⁵ cells/mL.
   NOTE 1: Cell density of 3*10⁵ cells/mL provides a seeding density of 15,000 cells/well. Seeding density is an important parameter and ideally should be pre-defined prior to the experiment. Seeding density should take into account 1) cell size – usually larger cells are seeded at a lower density; 2) treatment duration – cells are typically seeded at a lower density for experiments that will last longer, and 3) cell division rate – cells with a higher rate of division are seeded at a lower density. Specific examples of optimized seeding densities: K562 cells, larger, 24 h duration – 10,000 cells/well; MOLM-13 cells, moderate size, 24 h treatment – 15,000/well; MOLM-13 cells, 48 h treatment – 8,000/well; small healthy peripheral blood mononuclear cells (PBMCs), 24 h treatment – 50,000/well; primary AML cells, 24 h treatment – 15,000-20,000/well.
   NOTE 2: The presence of FBS in media may affect the activity of the compounds. Reducing the concentration of FBS may make assay results simpler to interpret, but it also reduces the physiological accuracy.
6. Seed 50 µL of cell suspension from step 1.5 into each well of a 96-well plate using a multichannel pipette.
7. Add compounds as follows:
   1. For single compound assays, add 50 µL of 2x compound solution into each well. For solvent-control wells, add 50 µL of test media containing the solvent at the 2x concentration.
   2. For combination assays, add 25 µL of each of the compounds (4x solutions) into each well. For single compound-control wells, add 25 µL of 4x compound solution and 25 µL of test medium. For solvent-control wells, add 50 µL of test medium or test medium containing the solvent.
      NOTE 1: The final concentration of DMSO should not exceed 0.5%.
      NOTE 2: It is recommended to add the media containing the compounds with the pipette touching the wall of each well due to low volume.
8. Gently tap the plate to ensure the mixing of the contents of the wells.
9. Incubate plates at 37 °C in a humidified 5% CO₂ atmosphere for an appropriate time, e.g., 24 h.

2. Cytotoxicity Assay: Staining with Hoechst 33342 and Propidium Iodide

1. Prepare 10x staining solution. This solution needs to be prepared fresh before each experiment, it cannot be stored. Final dye concentrations should be determined prior to the experiment.
   1. For leukemia cell lines and primary leukemia cells, 1 mL of 10x staining buffer contains 10 µL of 20 mM Hoechst 33342 and 50 µL of 1 mg/mL propidium iodide in sterile PBS (final concentrations: Hoechst 33342 20 µM, PI 5 µg/mL).
   2. For healthy PBMCs, 1 mL of 10x staining buffer contains 10 µL of 20 mM Hoechst 33342 and 10 µL of 1 mg/mL propidium iodide in sterile PBS (final concentrations: Hoechst 33342 20 µM, PI 1 µg/mL).
      NOTE: The final concentration of propidium iodide has to be determined prior to the experiments. Cells should be tested using a range of PI concentrations (1, 2.5, 5 µg/mL), and then Hoechst/PI-calculated viability should be compared with viability measured via trypan blue. The PI concentrations listed above were chosen based on target cell viability in media-control wells (above ~90% for leukemia cell lines, above ~70% for healthy PBMCs).
      CAUTION: Hoechst 33342 and propidium iodide are potential carcinogens. Wear appropriate personal protective equipment when handling them.
2. After incubation, use a multichannel pipette to add 10 µL of 10x staining buffer to each well.
   NOTE: To prevent cross-contamination, make sure that the pipette tips do not touch the media.
3. Gently tap the plate to mix and clear bubbles. Stain at 37 °C for 15 min.
4. Centrifuge the plate at 200 g for 4 min to bring all of the cells to the bottom of the plate. Carefully wipe the bottom of the plate with a damp kimwipe to remove fibers and/or debris that will interfere with imaging.
   NOTE 1: Centrifugation of the plate ensures the highest chances for all of the cells to be captured in the image. Often dead cells will detach and float, providing misleading values of cytotoxicity. Centrifugation mitigates this effect.
   NOTE 2: The plate should be imaged as quickly as possible after centrifugation, ideally, within 15 min. Centrifugation can reduce the selectivity of PI staining and may allow cells to slowly accrue propidium iodide. The improvement in accuracy gained by visualizing the dead cells outweighs the slight increase in PI staining