1. Culture cells in appropriate medium and vessel for microscopy
Two-color dual-parameter cell viability assay
This kit permits quick and easy determination of cell viability using two common microscope filters (FITC and RFP) based on intracellular esterase activity and plasma membrane integrity.
This protocol can be used for:
- Identifying live and 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
- LIVE/DEAD Viability/Cytotoxicity Kit (Cat. No. L3224)
- Dulbecco’s Phosphate-Buffered Saline (DPBS) (Cat. No. 14040-117)
- Fluorescence microscope with FITC and RFP filters
Protocol
2. Thaw vials
3. Add 5 µL calcein AM (Component A) and 20 µL ethidium homodimer-1 (Component B) to 10 mL DPBS to create staining solution.
4. Remove medium from cells
5. Add 100–200 µL of the staining solution directly to cells
6. Incubate 30 minutes at 20–25°C
7. Image cells
Spectral information and storage
Calcein AM | Ethidium homodimer-1 | |
---|---|---|
Excitation/emission | 494/517 nm | 528/617 nm |
Standard filter set | FITC or GFP | RFP |
EVOS Light Cube | GFP | RFP |
Storage conditions | –20°C | –20°C |
Protocol tips
- Use stock solutions within 1 day
- Optimal dye concentrations are likely to vary depending on cell type; use the highest dye concentration that gives minimal background
- The stains in this kit do not survive fixation or permeabilization
Kangaroo rat (PtK2) cells stained with the LIVE/DEAD Viability/Cytotoxicity Kit.