Preparing the Assay

• The protocol is simple to follow, and takes approximately 20 minutes to process at 37 degrees Celsius. The living cells used in the assay can take between several hours and several days to prepare for use. The control cells may take several hours to prepare, as they need to be induced to undergo apoptosis.
  
  

Protocol Part 1

• Culture cells to a density optimal for apoptosis or oxidative stress induction, according to your specific induction protocol, but not to exceed 10(6) cells/mL.
  
  

Protocol Part 2

• At the same time, culture a non-induced negative control cell population (at the same density as the induced population.
  
  

Protocol Part 3

• Induce apoptosis or purposely depolarize the mitochondria following your desired protocol.
  
  

Protocol Part 4

• Prepare 1X Assay/Wash Buffer.
  
  

Protocol Parts 5 to 8

• Prepare the 1 mM or 0.2 mM MitoPT™ TMRE/TMRM stock. For Protocol Part 6, prepare a 1:100 dilution of the 1 mM, or a 1:20 dilution of the 0.2 mM MitoPT™ TMRE/TMRM solution for spiking into cell culture samples.
  
  For Protocol Part 7, stain cells using a final 20-200 nM concentration of MitoPT™ TMRE/TMRM solution.
  
  For Protocol Part 8, analyze the data.