Cell Counting Kit-8 Plus KB1613-5ML

Product Information

Product Description/Introduction

This kit uses WST-8 compound, which can be reduced by dehydrogenase to form water-soluble orange formaldehyde compound in mitochondria, and the maximum absorption peak is 450nm. The production of formaldehyde is proportional to the number of living cells, that is, the more living cells, the darker the color; The higher the cytotoxicity, the lighter the color. For the same cells, there is a linear relationship between the depth of color and the number of cells.

Cell Counting Kit-8 Plus(CCK-8 Plus), is optimized on the basis of conventional CCK-8 kits and takes only 0.5 to 1 hour to complete the detection, with faster detection, higher sensitivity, and wider linear range.

Storage and Shipping Conditions

Ship with wet ice; store at 2-8℃ in the dark for up to 12 months, and -20℃ to 24 months.

Product Contents

Assay Protocol / Procedures

Cell Proliferation-Viability-Toxicity Assay

(1) In a 96-well plate, plant a certain amount of 100 µL cell suspension and pre-cultured in an incubator (37℃, 5% CO2) for 24 hours.

(2) 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, 48 or 72hours).

(3) Add 10 µL of CCK-8 plus 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 before adding CCK-8.

(4) Incubate the plate in the incubator for 0.5-2 hours.

(5) Measure the absorbance at 450 nm with a microplate reader.(wavelengths greater than 600nm, such as 650nm, can be used as a reference wavelength for dual-wavelength determination).

(6) If the OD value is not to be measured temporarily, you can add 10 µL of 0.1M 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.

Vitality Calculation

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

Cytotoxic viability (%)=[A (control)-A (dosed)]/[A (control)-A (blank)]×100%

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

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

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

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

3. 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.

4. 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.

5. Air bubbles in wells should be removed before the measurement of absorbance.

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