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HCS Mitochondrial Health Kit Protocol
Mitotoxicity and cytotoxicity cell health assay
The HCS Mitochondrial Health Kit simultaneously and quantitatively measures two important cell-health parameters: mitotoxicity and cytotoxicity. The MitoHealth stain detects changes in the mitochondrial membrane potential, whereupon the reagent accumulates in active mitochondria. The kit includes the Image-iT DEAD Green Stain to detect cytotoxicity, an impermeant dye to healthy cells that can enter the cells when the plasma membrane of cells is compromised.
This protocol can be used for:
- Identifying changes in the mitochondrial membrane potential
- Identifying dead cells using high-content imaging
This protocol should not be used for:
- Flow cytometry
You will need the following for this protocol:
- Cells growing on 96-well plates in culture
- Mitochondrial Health Kit (MitoHealth stain and Image-iT DEAD Green Stain) (Cat. No. H10295)
- Complete medium (e.g., for HeLa cells, Dulbecco's Modified Eagle Medium, Invitrogen (Cat. No. 11095-114))
- Phosphate-buffered saline (Cat. No. 14190-144)
- Paraformaldehyde 16% aqueous solution
- Fluorescence microscope with GFP, DAPI, and RFP filters
Protocol
 1. Add 30 μL DMSO to the tube of lyophilized MitoHealth stain to solubilize it (this step is needed the first time you use the kit only) |
 2. Culture cells in 100 µL per well of appropriate growth medium |
 3. Add 25 µL of test compound or drug to cells and incubate for the desired period of time under culture conditions |
 4. Prepare the cell staining solution by adding 10.5 μL of the MitoHealth stain (Component B) and 2.1 μL of Image-iT DEAD Green Stain (Component A) to 6 mL complete medium |
| 5. Add 50 µL of the cell staining solution per well and incubate for 30 minutes under culture conditions—total volume should be 175 µL at this point |
| 6. Add 3 mL 16% paraformaldehyde and 6 μL of Hoechst 33342 (Component C) to 9 mL PBS to create counterstain/fixation solution |
 7. Gently remove medium from each well |
 8. Add 100 µL of counterstain/fixation solution per well |
 9. Incubate for 15 minutes at room temperature |
 10. Gently remove counterstain/fixation solution |
 11. Wash wells once with 100 µL PBS |
| 12. Add 200 µL PBS |
 13. Image and analyze the results |
Protocol tips
- Allow cells to grow to 80% confluence before treatment
- Compatible with fixation, but not required
- Dye stock solutions should be stored frozen
- Thaw all kit components to room temperature before use
- DMSO concentrations higher than 0.5% should not be used with live cells
Spectral information
| Hoechst 33342 | Image-iT DEAD Green Stain | MitoHealth Stain |
|---|---|---|
| DAPI filter cube excitation 357/44 nm & emission 447/60 nm | GFP filter cube excitation 470/22 nm & emission 510/42 nm | RFP filter cube excitation 531/40 nm & emission 593/40 nm |