1. Remove the LIVE/DEAD® reagent stock solutions from the freezer and allow them to warm to room temperature.
  2. Add 5μl of and 15μl of 2mM Ethidium homodimer (EthD-1) stock solution to 10 mL of sterile, tissue culture–grade Dulbecco’s phosphate-buffered saline (D-PBS), vortexing to ensure thorough mixing.
  3. Combine the reagents by transferring 5 μL of the supplied 4 mM calcein AM stock solution to the 10 mL PI. Vortex the resulting solution to ensure thorough mixing.
  4. Note that aqueous solutions of calcein AM are susceptible to hydrolysis. Aqueous working solutions should therefore be used within one day.
  5. Wash the gels prior to the assay to remove or dilute serum esterase activity generally present in serum-supplemented growth media (serum esterases could cause some increase in extracellular fluorescence by hydrolyzing calcein AM). Wash gels gently with 1.2mL of Dulbecco’s phosphate-buffered saline (D-PBS)
  6. Add 100 – 300uL of stain onto the gel
  7. Incubate the gel for 30–45 minutes at room temperature. A shorter incubation time may be used if the dye concentrations or incubation temperature are increased.
  8. View the labelled cells under the fluorescence microscope.
  9. Calcein and PI 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. 494 nm (green, calcein) and 535 nm (red, propidium iodide) emission filters.