Cell Counting Kit-8 (CCK-8),IC-5885

For In Vitro Research Use Only

Manufactured by: InCellGen Inc.

1. Introduction

The Cell Counting Kit-8 (CCK-8) allows sensitive colorimetric assays for the determination of cell viability in cell proliferation and cytotoxicity assays. The kit utilizes a highly water-soluble tetrazolium salt, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2H-tetrazolium, monosodium salt], which is reduced by cellular dehydrogenases to an orange-colored formazan dye that is soluble in the tissue culture medium.

The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. The CCK-8 reagent is non-radioactive and safer than other tetrazolium salts like MTT, as it does not require a solubilization step.

2. Kit Components

CCK-8 Solution  5mL x 1 bottle |2-8¡æ, protected from light

*Note: One bottle (10 mL) is sufficient for 1,000 assays in a 96-well plate (i.e., 10 µL per well).*

3. Material Required but Not Supplied

-Cell culture plates (e.g., 96-well, 24-well)

-Cell culture medium

-Incubator (37¡æ, 5% CO₂)

-A microplate reader capable of measuring absorbance at 450 nm.

4. Storage and Stability

-Upon receipt, store the CCK-8 solution at 2-8¡æ in the dark.

-The kit is stable for 12 months from the date of receipt when stored properly.

-After thawing, mix the solution thoroughly before use. For convenience, the required amount of CCK-8 solution can be aliquoted to avoid repeated freeze-thaw cycles.

-If the solution turns a noticeable orange color, it may indicate contamination or degradation and should not be used.

5. Assay Procedure

5.1. Cell Seeding and Treatment

1. Seed cells in a 96-well culture plate at an appropriate density (e.g., 1,000 - 10,000 cells per well in 100 µL of culture medium). Include a background control well (medium only, no cells).

2. Pre-incubate the plate in a humidified incubator (37¡æ, 5% CO₂) for 24 hours or until the cells adhere.

3. Treat the cells with the test compounds at desired concentrations for the specified duration.

5.2. CCK-8 Addition and Incubation

1. At the end of the treatment period, add 10 µL of the CCK-8 solution directly to each well containing 100 µL of culture medium.

2. Gently swirl the plate to ensure thorough mixing.

3. Incubate the plate in the incubator (37¡æ, 5% CO₂) for 1-4 hours.

-  Optimization Note: The incubation time can vary depending on cell type, cell number, and metabolic activity. It is recommended to determine the optimal incubation time by monitoring the color development. The absorbance (OD 450 nm) should be within the linear range of your plate reader (typically between 0.5 and 2.0).

5.3. Absorbance Measurement

1. Measure the absorbance of each well at 450 nm using a microplate reader.

2. The reference wavelength can be set to 600-650 nm to subtract background signals from the plate and medium.

6. Data Analysis

Calculate the cell viability using the following formula:

Cell Viability (%) = [ (OD_Treatment-OD_Background)/(OD_Control - OD_Background) ] ¡Á 100%

- OD_Treatment: Absorbance of wells with cells and test compound.

- OD_Control: Absorbance of wells with cells and solvent/vehicle control.

- OD_Background: Absorbance of wells with medium and CCK-8 only (no cells).

7. Key Advantages

-  Highly Sensitive: More sensitive than MTT and other tetrazolium salts.

-  One-Step Procedure: Just add, mix, and incubate.

-  Non-Radioactive: Safe and convenient.

-  Water-Soluble: No solubilization step is required, saving time and reducing variability.

-  Biocompatible: The CCK-8 reagent is non-toxic to cells, allowing for continuous monitoring of the same sample over time.

8. Precautions & Troubleshooting

- Avoid introducing bubbles during pipetting, as they can interfere with absorbance readings.

- Protect the CCK-8 reagent from light during storage and use.

- The culture medium should be clear, as phenol red does not interfere with the assay at 450 nm.

- Low Signal: Increase the number of cells per well or extend the incubation time with CCK-8.

- High Background: Ensure the background control (medium only) is properly set. Reduce the incubation time if the signal is saturated.

9. Technical Support

For any questions or technical support, please contact:

InCellGen Inc.

Email: info@incellgene.com

Website: www.incellgene.com

This product is for research purposes only. It is not for diagnostic or therapeutic use.
10.Order information