The polyanionic dye calcein AM is well-retained within live cells, producing an intense uniform green fluorescence in live cells (excitation/emission ~495 nm/~515 nm), while ethidium homodimer-1 (EthD-1) enters cells with damaged membranes to produce a bright red fluorescence in dead cells (excitation/emission ~495 nm/~635 nm).
Protocols are provided for fluorescence microscopy or microplate analysis of adherent cells, or flow cytometry analysis of cells in suspension.
- Adherent or suspended NSCs
Media and Reagents
- LIVE/DEAD® Viability/Cytotoxicity Assay Kit (Cat. no. L-3224)
- Calcein AM
- Ethidium homodimer-1 (EthD-1)
- Dulbecco’s Phosphate-Buffered Saline (D-PBS) (Cat. no. 14040)
- Fluorescence microscope
Note: Calcein and EthD-1 can be viewed simultaneously with a conventional fluorescein longpass filter. The fluorescence from these dyes may also be observed separately; calcein can be viewed with a standard fluorescein bandpass filter and EthD‑1 can be viewed with filters for propidium iodide or Texas Red® dye.
- Remove the stock solutions provided in the kit from the freezer and allow them to warm to room temperature. Gibco® Neurobiology Protocols Handbook | 65 Cell Analysis
- Add 20 µL of the supplied 2 mM EthD-1 stock solution (Component B) to 10 mL of sterile, tissue culture–grade D-PBS. Vortex to ensure thorough mixing. This prepares a ~4 μM EthD-1 solution.
- Combine the reagents by adding 5 μL of the supplied 4-mM calcein AM stock solution (Component A) to the 10 mL of EthD-1 solution in D-PBS. Vortex the resulting solution to ensure thorough mixing.
Note: This reagent mixture is suitable for most neural cells. For cells with higher esterase activity, you might need to start with a lower calcein AM concentration. For further information, refer to the user manual provided with the LIVE/DEAD® Viability/Cytotoxicity Assay Kit.
The resulting working solution of ~2 μM calcein AM and ~4 μM EthD-1 is ready to be used. The final concentration of DMSO is ≤ 0.1%, a level generally innocuous to most cells.
Note: Prepare a freshly coated culture vessel each time before plating cells. There is no need to rinse the culture vessel before use.
Adherent NSCs may be cultured on sterile glass coverslips or in a multiwell plate.
- Aspirate the medium supernatant and wash the cells gently with the same volume of D-PBS prior to the assay to remove or dilute any serum esterase activity. Note: Serum esterases could cause some increase in extracellular fluorescence by hydrolyzing calcein AM.
- Fluorescence microscopy: Transfer an aliquot of the cell suspension to a coverslip and allow the cells to settle on the surface at 37°C in a covered petri dish. Then add 100–150 μL of prepared LIVE/DEAD® reagent to the coverslip, so that all cells are covered by solution. Microplate reader: Add an aliquot of the cell suspension to each microplate well in a sufficient volume to cover at least the bottom of each well. Then add an approximately equal volume of prepared LIVE/DEAD® reagent.
- Incubate the cells at room temperature for 10–30 minutes. Measure fluorescence using the appropriate excitation and emission filters.
- Analyze the sample under a fluorescence microscope or using a fluorescence microplate reader.
Determining Viability of Cells in Suspension with Flow Cytometry
Allow all the reagents to come to room temperature before proceeding.
- Make an 80-fold dilution of calcein AM (Component A) in DMSO to make a 50 μM working solution (e.g., add 2 mL of calcein AM to 158 mL DMSO).
- Prepare a 1-mL suspension of cells with 0.1 × 106 to 5 × 106 cells/mL for each assay. Cells may be in culture medium or buffer.
- Add 2 μL of a 50-μM calcein AM working solution and 4 μL of the 2-mM EthD-1 stock to each milliliter of cells. Mix the sample.
- Incubate the cells for 15–20 minutes at room temperature, protected from light.
- As soon as possible after the incubation period (within 1–2 hours), analyze the stained cells by flow cytometry using 488 nm excitation and measuring green fluorescence emission for calcein (i.e., 530/30 bandpass) and red fluorescence emission for EthD-1 (i.e., 610/20 bandpass).
- Gate on cells to exclude debris. Using single color-stained cells, perform standard compensation. The population should separate into two groups: live cells will show green fluorescence and dead cells will show red fluorescence (Figure 1).
Figure 1 - Flow cytometry viability assay using the LIVE/DEAD® Viability/Cytotoxicity Kit. A 1:1 mixture of live and ethanol-fixed human B cells was stained with calcein AM and EthD-1 following the protocol provided. Flow cytometry analysis was performed with excitation at 488 nm. The resulting bivariate frequency distribution shows the clear separation of the green fluorescent (530 nm) live cell population from the red fluorescent (585 nm) dead cell population.