Advantages

1. One-bottle, ready-to-use solution. 
2. The toxicity of CCK-8 is so low that, after the CCK-8 assay is completed, the same cells can be used for other cell proliferation assays. 
3. No harvesting, no washing and no solubilization steps 
4. CCK-8 has higher sensitivity than MTT or MTS

Protocol

1. Inoculate cell suspension (100 μl/well) in a 96-well plate. Also prepare wells that contain known numbers of viable cells (to create a calibration curve in step 5). Pre-incubate the plate in a humidified incubator (e.g., at 37 °C, 5% CO2). 
2. Thaw the CCK-8 on the bench top if it is frozen.
   Note: It takes about 30 minutes on the bench top at 25 °C. 
3. Add 10 μl of the CCK-8 solution to each well of the plate.
   Note: Be careful not to introduce bubbles to the wells, since they interfere with the O.D. reading. 
4. Incubate the plate for 1-4 hours in the incubator. 
5. Measure the absorbance at 450 nm using a microplate reader. Prepare a calibration curve using.

Precautions

1. Repeated thawing and freezing causes an increase in the background, which interferes with the assay.
2. Keep CCK-8 protection from light all the time.

Background control

• Slight spontaneous absorbance around 450 nm occurs in culture medium incubated with CCK-8. This background absorbance depends on the culture medium, pH, incubation time and length of exposure to light. Typical background absorbance after 2 hours incubation is 0.1 - 0.2 absorbance units. To correct for this, prepare one or more control wells without cells, and subtract the average absorbance of the control wells from that of the other wells.

Additional information