LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells

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

1. Culture cells in appropriate medium and vessel for microscopy

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.

LIVE/DEAD Viability/Cytotoxicity Kit for Mammalian Cells Protocol