Enhanced Cell Counting Kit-8

General Information

Enhanced Cell Counting Kit- 8 (CCK-8) allows convenient, one-step cell counting by the reduction reaction of WST-8, a highly water-soluble tetrazolium salt. In the presence of dehydrogenases and an electron carrier, WST-8 produces a yellow-colored formazan dye which can be quantified by microplate reader. The amount of formazan dye generated by a sample culture is directly proportional to the number of viable cells, allowing for sensitive quantification of cell proliferation and cytotoxicity assays.

Advantages

  • Low toxicity to cells
  • One-bottle, no premixing of reagents
  • No organic solvents or isotopes required
  • No harvesting, no washing and no solubilization steps
  • More sensitive than MTT, XTT, MTS or WST-1

Storage

Store the kit at 4°C away from light for one year

General Protocol

Cell Number Determination

  1. Collect cells needed for the experiment and prepare wells that contain known numbers of viable cells.
  2. Pre-incubate adherent cells. and add 10 μl of the CCK-8 solution to each well.
  3. Incubate the plate for 1-4 hrs in the incubator.
  4. Measure the absorbance at 450 nm using a microplate reader. Prepare a calibration curve using the data obtained from the wells that contain known numbers of viable cells.
  5. Under consistent condition, the cell number of the unknown sample can be calculated according to the standard curve.

Cell Viability Assay

  1. Inoculate cell suspension (above 5000 cells /100 μl/ well) in a 96-well plate. The cells for the assay should enter into the logarithmic growth phase. The average incubation time to enter this phase is from 24 hrs to 48 hrs. Please check cell databases to estimate the pre-incubation time.
  2. Add 10μl of the CCK-8 solution to each well of the plate. Be careful not to introduce bubbles to the wells.
  3. Incubate the plate for 1-4 hrs in the incubator. The absorbance will differ among cell types even if the number of cells/well and coloration time are the same. Set an appropriate incubation time to give a proportional relationship between the cell number and the absorbance.
  4. Measure the absorbance at 450 nm using a microplate reader. To measure the absorbance later on, add 10μl of 1% w/v SDS or 0.1 M HCl to each well, cover the plate and store it with away from light at room temperature.

Cell Proliferation and Cytotoxicity Assay

  1. Dispense 100μl of cell suspension in a 96-well plate at a density of 5*103-105 cells/well. Pre-incubate the plate overnight in a humidified incubator.
  2. Add various concentrations of toxicant into the culture media in the plate.
  3. Incubate the plate for an appropriate length of time (e.g., 3, 6, 12, 24 or 48 hrs) in the incubator.
  4. Add 10 μl of CCK-8 solution to each well of the plate. Be careful not to introduce bubbles in the wells.
  5. Incubate the plate for 1-4 hrs in the incubator. The incubation time varies by the type and number of cells in a well.
  6. Measure the absorbance at 450 nm using a microplate reader. To measure the absorbance later on, add 10μL of 1% w/v SDS 0.1 M HCl to each well, cover the plate and store it away from light at room temperature.