MitoSpy™ Orange and MitoSpy™ Red contain a chloromethyl group that covalently attaches to cysteines in the cell. This gives them a higher probability of remaining in a cell after the sample is fixed and permeabilized. However, as the fix/perm process washes out a large amount of the reagent, a much higher concentration of MitoSpy™ Orange and MitoSpy™ Red is required to get sufficient staining.

After the fix/perm process, most MitoSpy™ Green and MitoSpy™ NIR will be washed away. As such, they are not recommended to be used with samples that require fixation and permeabilization.

*Note that all MitoSpy™ products must be used on live samples. The recommendations below reflect whether MitoSpy™ is successfully retained following the "Retention Condition" treatment.

Retention Condition* Recommended MitoSpy™ Orange Concentration Recommended MitoSpy™ Red Concentration Recommended MitoSpy™ Green Concentration Recommended MitoSpy™ NIR Concentration
Live Cells 50-250 nM 50-250 nM 50-250 nM 1-50 nM
Fixed Cells 50-250 nM 50-250 nM 50-250 nM Not recommended.
Fixed and Permeabilized Cells 250-500 nM 250-500 nM Not recommended. Not recommended.
MitoSpy™ Orange Live staining (50 nM)
PFA-fixed and permeabilized staining (500 nM)
MitoSpy™ Green

HeLa cells that were stained live with either MitoSpy™ Orange (yellow) or Green (green). Cells that were fixed and permeabilized with 4% PFA and 0.1% Triton X-100 were also stained with DAPI (blue). Photos were taken with a 60x magnification.