CytoFix™ Red Mitochondrial Stain

1. Prepare cells in growth medium
2. Incubate cells with CytoFix™ MitoRed working solution for 20-30 minutes at 37 °C
3. Remove CytoFix™ MitoRed working solution
4. Fix cells with formaldehyde (Optional)
5. Analyze under fluorescence microscope with Cy3/TRITC filter set

For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Add 20 µL of the stock solution into 10 mL of Hanks and 20 mM Hepes buffer (HHBS) or buffer of your choice or cell culture medium, and mix well.

Note: 20 µL stock solution is enough for one 96-well plate assay. The staining conditions may be modified according to the particular cell type and the permeability of the cells or tissues to the probe.

Note: Unused CytoFix™ MitoRed stock solution can be aliquoted and stored at ≤ -20 °C with smaller aliquots. Protect from light and avoid repeated freeze-thaw cycles.

1. Prepare cells in growth medium.

2. Add 100 µL/well (96-well plate) or 50 µL/well (384-well plate) of CytoFix™ MitoRed working solution in the cell plate.

   Note: The optimal concentration of the cell membrane probe varies depending on the specific application.

   Note: Serum in the culture growth medium may interfere with the staining. Staining in the absence of a culture growth medium should improve the staining intensity.

3. Incubate the cells at 37 °C for 20-30 minutes, protected from light.
4. Remove working solution in each well. Wash cells with HHBS or buffer of your choice. (Optional)

5. Optional: Fix the cells with a 4 % solution of paraformaldehyde for 20-30 minutes at room temperature. Wash twice to get rid of the fixation solution.

6. Observe the fluorescence signal in cells using fluorescence microscope with a Cy3/TRITC filter set.