Cell Meter™ Fluorimetric Cell Cytotoxicity Assay Kit

Protocol summary

1. Prepare cells with test compounds (100 µL/well/96-well plate or 50 µL/well/384-well plate)
2. Add 1/5 volume of Assay Solution (Component A)
3. Incubate the cells in a 37°C, 5% CO2 incubator for 1 - 24 hours 
4. Monitor fluorescence intenstity (bottom read mode) at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important notes
Thaw the component at room temperature before starting the experiment.

1. Plate 100 to 10,000 cells per well in a tissue culture microplate with black wall and clear bottom. Add test compounds into the cells, and incubate for a desired period of time (such as 24, 48 or 96 hours) in a 37°C, 5% CO₂ incubator. For blank wells (medium without the cells), add the same amount of compound buffer. The suggested total volume is 100 µL for a 96-well plate, and 50 µL for a 384-well plate.
   
   
2. Set up the following controls at the same time.
   
   1. Positive control: contains cells and known proliferation or cytotoxicity inducer.
      
      
   2. Negative control: contains cells but no test compounds.
      
      
   3. Vehicle control: contains cells and the vehicle used to deliver test compounds.
      
      
   4. Non-cell control: contains growth medium without cells. Note: LDH in serum will contribute to background fluorescence.
      
      
   5. Test compound control: contains the vehicle used to deliver test compounds [Hank’s balance salt solution (HBSS) or phosphate-buffered saline (PBS)] and test compound. Some test compounds have strong autofluorescence and may give false positive results. Note: Match the total volume of all the controls to 100 µL for a 96-well plate or 50 µL for a 384-well plate with growth medium.
      
      
3. Warm up the Assay Solution (Component A) to 37°C, and mix it thoroughly before starting the experiments.
   
   
4. Add 20 µL/well (96-well plate) or 10 µL/well (384-well plate) of Assay Solution (Component A) into each well. Mix the reagents by shaking the plate gently for 30 seconds.
   
   
5. Incubate the cells in a 37°C, 5% CO₂ incubator for 1 - 24 hours, protected from light. Note: The appropriate incubation time depends on the metabolism rate of the individual cell type and cell concentration used. Optimize the incubation time for each experiment. Extremely prolonged incubation time is not recommended since resazurin could be converted to colorless dihydroresorufin.
   
   
6. Monitor the fluorescence intensity with a fluorescence microplate reader (bottom read mode) at Ex/Em = 540/590 nm (Cutoff = 570nm).