HCS Mitochondrial Health Kit Protocol | Thermo Fisher Scientific

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