1. Culture cells in appropriate medium and vessel for microscopy
Membrane integrity–based viability assay
NucRed Dead 647 ReadyProbes Reagent is a cell-impermeant stain that emits bright far-red fluorescence when bound to DNA. Since this reagent stains cells that lack plasma membrane integrity, it is extremely useful to measure viability and to measure cytotoxicity in kinetic live-cell assays. NucRed Dead 647 reagent is also suitable for staining nuclei in fixed-cell preparations and tissue sections.
This protocol can be used for:
- Identifying dead cells using a fluorescence microscope
This protocol should not be used for:
- Flow cytometry
You will need the following for this protocol:
- Cells growing in culture
- NucRed Dead 647 ReadyProbes Reagent (Cat. No. R37113)
- Fluorescence microscope with far red or Cy5 filters
Protocol
2. Add 2 drops NucRed® Dead 647 reagent per milliliter of medium to label cells
3. Incubate 5–15 minutes
4. Image cells
Spectral information and storage
| NucRed Dead 647 ReadyProbes Reagent | |
|---|---|
| Excitation/Emission | 642/661 nm |
| Standard filter set | Cy5 |
| EVOS Light Cube | Cy5 |
| Storage conditions | Room temperature |
Protocol tips
- In some cases, more or fewer drops may be needed to achieve optimal staining intensity
- Image quality may be improved by replacing medium with Live Cell Imaging Solution (A14291DJ)
Tubulin and nuclear staining in HeLa cells. HeLa cells were fixed and permeabilized and stained for tubulin (biotinylated mouse anti-a-tubulin mouse monoclonal antibody followed by an Alexa Fluor 488 goat anti-mouse secondary antibody and counter-stained with NucRed Dead 647 ReadyProbes reagent.