Cell Meter™ Colorimetric 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 absorbance ratio at A_570nm/A_605nm 

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 corresponding 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.
      
      
   5. Test compound control: contains the vehicle control 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. Mix it thoroughly before starting the experiments.
   
   
4. Add 20 µL (96-well plate) or 10 µL (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 the indicator could be converted to colorless compound.
   
   
6. Monitor the absorbance increase with an absorbance plate reader at OD ratio A_570nm/A_605nm. The ratio of OD₅₇₀ to OD₆₀₅ is used to determine the cell viability in each well. Note: The cell viability is proportional to increased OD₅₇₀ and decreased OD₆₀₅.