Cell Proliferation - Toxicity Assay Kit

Cell Counting Kit-8 (CCK-8)    

Product Introduction:

CCK-8 kit is a rapid and highly sensitive detection kit based on WST-8 that is widely used for cell proliferation and cytotoxicity. WST-8 is a compound similar to MTT . In the presence of the electron coupling reagent 1-Methoxy PMS , it can be reduced to generate orange-yellow water-soluble formazan . The more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of color is linearly related to the number of cells.

Directions:

1. Inoculate the cell suspension in a 96-well plate ( 100 ul/ well ) . Usually, there are about 2,000 cells per well for cell proliferation experiments and about 5,000 cells per well for cytotoxicity experiments . The specific number of cells used in each well depends on factors such as cell size and cell proliferation rate .

2. According to the experimental needs, culture and give 0-10ul specific drug stimulation for an appropriate period of time ( for example : 6, 12, 24 or 48 hours ) .

3. Add 10ul CCK-8 solution to each well . If the initial culture volume is 200ul , then add 20ul CCK-8 solution, and so on. You can use the wells with the corresponding amount of cell culture medium and CCK-8 but no cells as blank controls. If you are worried that the drugs used will interfere with the test, you need to set up the wells with the corresponding amount of cell culture medium, drugs and CCK-8 solution but no cells as blank controls.

4. Continue incubating in the cell culture incubator for 1-4 hours. The specific time can be determined through preliminary experiments. In the preliminary experiment, the microplate reader can be used to detect after 0.5 , 1 , 2 and 4 hours, and then a time point with a more suitable absorbance range can be selected for subsequent experiments.

5. Use an ELISA reader to measure the absorbance at 450 nm. If there is no 450 nm filter, a 420-480 nm filter can be used. If the sample is a highly turbid cell suspension, a wavelength greater than 600 nm , such as 650 nm , can be used as a reference wavelength for dual wavelength measurement.

6. If you do not need to measure the OD value temporarily, you can add 10ul of 0.1M HCl solution or 1% w/v SDS solution to each well and store it at room temperature away from light . The absorbance will not change within 24 hours.

Note:

1. When using a 96-  well plate for testing, if the cell culture time is long, please pay attention to the evaporation problem. You can add culture medium, water or PBS  to the outer circle of the 96-  well plate to keep it moist. At the same time, you can place the 96-  well plate near the water tray in the incubator to alleviate evaporation.

2. The culture time varies according to the cell type and the number of cells in each well. Before the formal experiment, it is recommended to do a preliminary experiment to explore the number of cells to be plated and the culture time after adding CCK-8  reagent.

3. When spreading the plates, please ensure that the number of cells in each well is uniform. It is recommended to mix the plates frequently during the spreading process to prevent unevenness caused by cell precipitation. After adding CCK-8,  please gently shake the culture plate several times to fully mix the culture medium and CCK-8  solution.

4. The detection of this kit depends on the reaction catalyzed by dehydrogenase. If the substance to be tested is oxidative or reductive, fresh culture medium can be replaced before adding CCK-8  to remove the influence of the drug. If the influence of the drug is relatively small, the culture medium can be left unchanged and the blank absorption after adding the drug to the culture medium can be directly deducted.

5. When adding CCK-8  , if the cell culture time is long and the culture medium color or pH  value has changed, it is recommended to replace it with fresh culture medium.

6. Before using the ELISA reader for testing, make sure there are no bubbles in each well, otherwise it will interfere with the measurement.

7. This kit is produced by aseptic filling. During use, please operate aseptically in a biological safety cabinet to avoid contamination.

8. For your health and safety, please wear a lab coat and disposable gloves or latex gloves when operating.

How to save:

    Store at 4 ℃ , valid for one year. For long-term storage, it is recommended to store at -20 ℃ , but do not freeze and thaw repeatedly.