TO-PRO-3 is a cell-impermeant dye that gives strong and selective staining of the nucleus in cultured cells and paraffin sections. TO-PRO-3 and other carbocyanine monomers have very strong binding affinity for dsDNA, with dissociation constants in the micromolar range. The long-wavelength fluorescence of TO-PRO-3 is outside the spectral range of tissue autofluorescence as well as commonly used fluorophores in the DAPI and FITC channels.
First, fix and permeabilize cultured cells with a protocol appropriate for your sample.
1. Wash the cells 1–3 times in PBS as needed.
2. Prepare the TO-PRO-3 staining solution by diluting the TO-PRO-3 stock solution 1:1,000 (1 µM) in PBS.
|3. Add sufficient staining solution to cover the cells.|
|4. Incubate for 15–30 minutes, protected from light.|
|5. Remove the staining solution.|
|6. Wash the cells 3 times in PBS.|
|7. Image the cells.|
|Standard filter set||Far Red|
|EVOS Light Cube||Cy®5|