Live/Dead Analysis Protocol

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Live/dead analysis protocol 3d bioprinted bioprinting


Follow these step-by-step instructions to perform a Live/Dead analysis on your 3D bioprinted constructs. This protocol uses a fluorescent assay to determine the number of viable cells in culture.


  • LIVE/DEAD Kit (Life Technologies #L-3224)
    • Calcein AM 4mM in DMSO
    • Ethidium homodimer 2 mM in DMSO/H2O 1:4
  • 15 mL conical tube
  • Aluminum foil
  • Sterile Phosphate Buffered Saline (PBS)
  • Vortex
  • Biosafety Cabinet
  • Pipet
  • Incubator
  • Water Bath


  1. Using a water or bead bath, warm reagents to 37˚C;
  2. Calculate how much assay solution you will need for your samples. Refer to the table below to check working volumes for different well plates:
Well PlateWell Working Volume
96-well200 µL
48-well300 µL
24-well500 µL
12-well1 mL
6-well2 mL
30 mm2 mL
60 mm3 mL
100 mm5 mL
Table 1: Well plate working volumes.
  1. Add 10 mL of sterile PBS to a 15 mL conical tube;
    1. Note: multiply quantities accordingly depending on the amount of solution determined on step 2. Always make at least 10% extra solution to ensure all wells will get the same amount of each component.
  2. Add 20 uL of the supplied 2 mM of ethidium homodimer (EthD-1) stock solution to the 10 mL of sterile PBS;
  3. Vortex this solution;
  4. Add 5 μL of the supplied 4 mM calcein-AM stock solution to the 10 mL EthD-1 solution;
  5. Vortex this solution;
  6. Wrap the tube in tin foil to keep it away from light. The resulting approximately 2 μM calcein AM and 4 μM EthD-1 working solution is then added directly to cells. The final concentration of DMSO is ≤ 0.1%, a level generally innocuous to most cells. This working solution is good for 1 day;
  7. In a biosafety cabinet, remove cell media from your dish that contains either cells or bioprinted constructs;
    1. Note: make sure to have your biosafety cabinet light off.
  8. Wash the constructs or cells 2x with sterile PBS to remove all media components;
  9. Add the appropriate volume of working solution to your wells according to the working volumes on step 2;
  10. Wrap the well plate in tin foil to protect it from light;
  11. Incubate it at 37˚C for 30-45 minutes. For hydrogels, incubate for 60 minutes;
  12. Remove working solution from the wells;
  13. Wash the tissues or cells 3x with PBS to stop the reaction;
  14. Image cells or constructs using a fluorescent or confocal microscope.

We hope this protocol will help you easily perform your live/dead analyses! For more bioprinting protocols – click here.

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