Live/Dead Analysis Protocol

Introduction

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.

Materials

• 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

Methods

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 Plate	Well Working Volume
96-well	200 µL
48-well	300 µL
24-well	500 µL
12-well	1 mL
6-well	2 mL
30 mm	2 mL
60 mm	3 mL
100 mm	5 mL

3. 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.
4. Add 20 uL of the supplied 2 mM of ethidium homodimer (EthD-1) stock solution to the 10 mL of sterile PBS;
5. Vortex this solution;
6. Add 5 μL of the supplied 4 mM calcein-AM stock solution to the 10 mL EthD-1 solution;
7. Vortex this solution;
8. 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;
9. 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.
10. Wash the constructs or cells 2x with sterile PBS to remove all media components;
11. Add the appropriate volume of working solution to your wells according to the working volumes on step 2;
12. Wrap the well plate in tin foil to protect it from light;
13. Incubate it at 37˚C for 30-45 minutes. For hydrogels, incubate for 60 minutes;
14. Remove working solution from the wells;
15. Wash the tissues or cells 3x with PBS to stop the reaction;
16. 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.