Cell Counting Kit-8 (CCK-8) provides a tool for studying induction and inhibition of cell proliferation in any in vitro model. Cell Counting Kit-8 (CCK-8) allows very convenient assays by utilizing highly water-soluble tetrazolium salt. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium, monosodium salt] produces a water-soluble formazan dye upon reduction in the presence of an electron mediator.

CCK-8 is a one-bottle solution, ready to use. CCK-8, being nonradioactive, allows sensitive colorimetric assays for the determination of the number of viable cells in cell proliferation and cytotoxicity assays.

1. More Sensitive than MTT, MTS, or WST-1

2. No Toxicity to Cell

3. Simple Steps (No Thawing Necessary)

4. Stable One Bottle Solution : 1 year at 4°C

Storage

Stored at 4°C protecting from light, and stable for up to 12 months.

Stored at -20°C protecting from light, and stable for up to 2 years.

Protocol

Cell Number Determination

1. Inoculate cell suspension (100 μL/well) in a 96-well plate. Pre-incubate the plate in a humidified incubator (e.g., at 37°C, 5% CO₂).

2. Add 10 μL of the CCK-8 solution to each well of the plate. Be careful not to introduce bubbles to the wells, since they interfere with the O.D. reading.

3. Incubate the plate for 1-4 hours in the incubator.

4. Measure the absorbance at 450 nm using a microplate reader.

Cell Proliferation and Cytotoxicity Assay

1. Seed cells in a 96-well plate at a density of 10⁴-10⁵ cells/well in 100 μL of culture medium with or without compounds to be tested. Culture the cells in a CO₂ incubator at 37°C for 24 hours.

2. Add 10 μL of various concentrations of substances to be tested to the plate.

3. Incubate the plate for an appropriate length of time (e.g., 6, 12, 24 or 48 hours) in the incubator.

4. Add 10 μL of CCK-8 solution to each well of the plate using a repeating pipettor. Be careful not to introduce bubbles to the wells, since they interfere with the O.D. reading.

5. Incubate the plate for 1-4 hours in the incubator.

6. Before reading the plate, it is important to mix gently on an orbital shaker for 1 minute to ensure homogeneous distribution of color.

7. Measure the absorbance at 450 nm using a microplate reader.