Cell Viability Assays for Neural Stem Cells

The LIVE/DEAD Viability/Cytotoxicity Assay Kit provides a two-color fluorescence cell viability assay that is based on the simultaneous determination of live and dead neural stem cells (NSCs) with probes that measure two recognized parameters of cell viability: intracellular esterase activity and plasma membrane integrity.

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).

• Adherent or suspended NSCs
• LIVE/DEAD Viability/Cytotoxicity Assay Kit, for mammalian cells
  • Calcein AM
  • Ethidium homodimer-1 (EthD-1)

  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.

  • Dulbecco’s Phosphate-Buffered Saline (DPBS)
  • Fluorescence microscope

Prepare the reagents in the LIVE/DEAD Viability/Cytotoxicity Assay Kit as follows:

1. Remove the stock solutions provided in the kit from the freezer and allow them to warm to room temperature.
2. Add 20 µL of the supplied 2 mM EthD-1 stock solution (Component B) to 10 mL of sterile, tissue culture-grade DPBS. Vortex to ensure thorough mixing. This prepares a ~4 μM EthD-1 solution.
3. 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 DPBS. Vortex the resulting solution to ensure thorough mixing.

Adherent NSCs may be cultured on sterile glass coverslips or in a multiwell plate.

1. 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.

1. 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.
2. Incubate the cells at room temperature for 10–30 minutes. Measure fluorescence using the appropriate excitation and emission filters.
3. Analyze the sample under a fluorescence microscope or using a fluorescence microplate reader.

Allow all the reagents to come to room temperature before proceeding.

1. 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).
2. 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.
3. 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.
4. Incubate the cells for 15–20 minutes at room temperature, protected from light.
5. 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).
6. 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).