Cell Counting Kit-8 (CCK-8 kit)

Product Information

Introduction

Cell Counting Kit-8, abbreviated as CCK-8 kit or CCK8 kit, provides a rapid and highly sensitive method for measuring cell proliferation and cytotoxicity. The detection principle is that, in the presence of electronically coupled reagents, the WST-8 compound in the detection reagent is reduced by intracellular dehydrogenase to a water-soluble orange-yellow dirty compound, which has a maximum absorption peak at 450 nm. The amount of methyl filth is proportional to the number of living cells, i.e., the more and faster the cells proliferate, the darker the color; the more cytotoxic the cells are, the lighter the color. For the same cells, there is a linear relationship between color intensity and cell numbers. The WST-8 has no obvious toxicity to cells. The WST-8 in this kit has no obvious toxicity to cells, and can be used for the detection of cell proliferation induced by exogenous cytokines, as well as the detection of cytotoxicity induced by drugs and other cytotoxic reagents, or the detection of cell growth inhibition induced by some drugs. It is an upgraded version of the MTT detection method.

The kit is easy to use, no need to prepare or dilute, no need to collect or wash the cells, no need to re-solubilize the methyl dirty compounds, and it is suitable for both adherent and suspension cells. Phenol red and serum have no significant effect on the determination of this kit.

Storage and Shipping Conditions

Ship with wet ice; store at 2-8 for up to 12 months, and -20 for up to 24 months if not used for a long time. Long-term storage is recommended to keep away from light.

Product Contents

Assay Protocol / Procedures

1. Make a Standard Curve (Perform if measuring the exact number of cells is required)

A) Count the number of cells in the prepared cell suspension with a cytometer then seed the cells.

B) Proportionally dilute the medium in equal proportions to form a cell concentration gradient. Generally, 3-5 cell concentration gradients are required, with 3-6 duplicate wells in each group.

C) Culture until the cells adhere to the wall, and then add CCK-8 reagent for a certain period of time to measure the OD value, and make a standard curve with the number of cells as the abscissa (X axis) and the OD value as the ordinate (Y axis). According to this standard curve, the number of cells in the unknown sample can be determined (the premise of using this standard curve is that the experimental conditions should be consistent, and it is convenient to determine the number of cells inoculated and the incubation time after adding CCK-8).

2. Cell Viability Assay

A) Seed cell suspension (100 µL/well) in a 96-well plate. Pre-incubate the plate in an incubator at 37°C with 5% CO2).

B) Add 10 µL of CCK-8 solution to each well (be careful not to create air bubbles in the wells, which will affect the OD value).

C) Incubate the culture plate in the incubator for 2 h.

D) Measure the absorbance at 450 nm with a microplate reader.

Note: If the OD value is not to be measured immediately, you can add 10 µL of 0.1 M HCl or 1% SDS (W/V) solution to each well, and cover the culture plate and store it at room temperature in the dark. The absorbance did not change within 24 h.

3. Cell Proliferation-Toxicity Assay

A) Prepare 100 µL of cell suspension in a 96-well plate. Pre-incubate the plates for 24 hours in an incubator at 37°C with 5% CO2).

B) Add 10 µL of different concentrations of the test compounds to the culture plate. Incubate for an appropriate period of time (eg: 6, 12, 24 or 48 hours).

C) Add 10 µL of CCK-8 solution to each well (be careful not to create air bubbles in the wells, which will affect the OD reading).

Note：If the test compounds is oxidative or reducing, the effect of the drug can be removed by replacing the medium with fresh medium (removing the medium, washing the cells twice with medium, then adding new medium) before adding CCK-8.

D) Incubate the plate in the incubator for 2 hours.

E) Measure the absorbance at 450 nm with a microplate reader.

F) If the OD value is not to be measured temporarily, if you plan to measure it later, you can add 10 µL of 0.1 M HCl or 1% SDS (W/V) solution to each well, and cover the culture plate and store it at room temperature in the dark. The absorbance does not change within 24 hours.

4. Vitality Calculation

Cell viability (%) = [A (dosed) - A (blank)] / [ A (0 dosed) - A (blank)] × 100

A (dosed): Absorbance of wells with cells, CCK-8 solution, and drug solution

A (blank): absorbance of wells with medium and CCK-8 solution without cells

A (0 dosing): Absorbance of wells with cells, CCK-8 solution and no drug solution

Cell viability: cell proliferative viability or cytotoxic viability

Note

1. This kit employs the reduction reaction catalyzed by dehydrogenase. Reducing agents or antioxidants that may interfere with the assay should be removed from samples to be tested.

2. It is recommended to perform pre-experiment to explore the number of inoculated cells and the incubation time after adding CCK-8 reagent.

3. Leukocytes may need to be cultured for a longer period of time.

4. When using standard 96-well plates, the minimum seeding volume of adherent cells is at least 1,000 cells / well (100 µL of medium). The sensitivity for detecting leukocytes is relatively low, it is recommended that inoculate at least 2,500 cells / well (100 µL of medium). If you use a 24-well or 6-well plate, first calculate the corresponding inoculum amount of per well and add CCK-8 solution at 10% of the total volume of medium in each well.

5. If a 450 nm filter is not available, a filter with absorbance between 430-490 nm can be used, but 450 nm has the highest detection sensitivity.

6. The absorbance of phenol red in the medium can be eliminated by subtracting the absorbance of the background in the blank well during calculation, so it will not affect the detection.

7. Air bubbles in wells should be removed before the measurement of absorbance, as this may interfere with the test results.

8. For your safty and health, please wear safety glasses, gloves, or protective clothing.

For Research Use Only!

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