Cell Counting Kit-8

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Cell Number Determination

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

2. Add 10 uL 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 uL of culture medium with or without compounds to be tested. Culture the cells in a CO2 incubator at 37C for 24 hours.

2. Add 10 uL 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 uL 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.