AffiASSAY® Cell Counting Kit-8

Key Features

• Rapid and sensitive: Provides accurate results within a short incubation period.
  Wide applicability: Compatible with a variety of cell types, including adherent and suspension cells.
• High throughput: Suitable for high-throughput screening applications.
• Versatile: Can be used for cell proliferation assays, cytotoxicity assays, and drug screening assays.
• Stable signal: Offers stable and reproducible results over a wide range of cell densities and experimental conditions.
  

Assay Principle

The AffiASSAY® CCK-8 kit uses the water-soluble tetrazolium salt WST-8, which is reduced by cellular dehydrogenases in viable cells to form a water-soluble formazan dye. The intensity of the orange-colored formazan dye produced is directly proportional to the number of living cells in the sample. The absorbance of the formazan dye can be measured spectrophotometrically at a wavelength of 450 nm using a microplate reader.  

Applications

• Cell viability assays: Determine the number of viable cells in culture.
  
• Cell proliferation assays: Assess the rate of cell proliferation and growth.
  
• Cytotoxicity assays: Evaluate the cytotoxic effects of compounds or treatments on cells.
  
• Drug screening assays: Screen potential drug candidates for their effects on cell viability and proliferation. 
  

Kit Contents

• CCK-8 reagent: WST-8 tetrazolium salt solution
• User manual: Detailed instructions for assay setup and data analysis

Precautions

• For research use only. Not for diagnostic or therapeutic purposes.
• Handle all reagents with care and follow the instructions provided in the user manual.
• Avoid exposure of reagents to light during storage and assay procedures.
• Use appropriate safety precautions when working with cell cultures and laboratory chemicals.

Materials Needed

• AffiASSAY® Cell Counting Kit (CCK-8)
  
• Cell culture medium
  
• Cells of interest
  
• 96-well microplate
  
• Pipettes and tips
  
• Microplate reader capable of measuring absorbance at 450 nm

Protocol

Prepare Cell Culture

• Culture cells of interest according to standard protocols in appropriate culture vessels until they reach the desired confluence or growth phase.

Prepare Working Solution

• Thaw the CCK-8 reagent and allow it to equilibrate to room temperature.
  
• Prepare the CCK-8 working solution by diluting the CCK-8 reagent with fresh cell culture medium at a ratio of 1:10 (e.g., 10 μL of CCK-8 reagent + 90 μL of medium per well). Prepare enough working solution for all wells to be assayed.

Seed Cells in Microplate

• Aspirate the culture medium from the cells and wash once with phosphate-buffered saline (PBS) if necessary.
  
• Add the appropriate volume of fresh cell culture medium containing cells to each well of a 96-well microplate. Adjust the cell density according to the experimental requirements.

Incubate Cells with CCK-8 Reagent

• Incubate the microplate containing cells with the prepared CCK-8 working solution. Ensure each well is evenly covered with the solution.
• Incubate the microplate at standard cell culture conditions (e.g., 37°C, 5% CO2) for a specified period of time (typically 1-4 hours). The optimal incubation time may vary depending on the cell type and experimental conditions.

Measure Absorbance

• After the incubation period, gently mix the contents of each well by pipetting up and down or using a plate shaker.
  
• Measure the absorbance of each well at 450 nm using a microplate reader. Ensure to use a reference wavelength if necessary according to the manufacturer's instructions.

Data Analysis

• Record the absorbance values for each well.
  
• Calculate the cell viability or proliferation using the formula provided by the manufacturer or based on the specific experimental design.
  
• Analyze the data and interpret the results accordingly.

Quality Control

• Perform appropriate controls, including background controls (no cells) and positive controls (e.g., untreated cells or cells treated with known compounds).
• Ensure all reagents are handled and stored properly according to the manufacturer's instructions to maintain assay reliability.

Troubleshooting

• If unexpected results are obtained, troubleshoot the assay by checking experimental conditions, reagent handling, and protocol adherence.
• Consult the user manual or contact technical support for further assistance if needed

Dispose of Reagents

• Dispose of all reagents and materials according to local regulations for biohazardous waste or chemical waste disposal.