Cholesterol plays a crucial role in various physiological processes, including the composition of cell membranes, hormone production, and cell signaling. As a vital component of animal cell structure, cholesterol is essential for maintaining membrane permeability and fluidity, as well as contributing significantly to cell signaling pathways. Monitoring cholesterol levels is critical for diagnosing and classifying hyperlipoproteinaemias, as it can provide valuable insights into liver and biliary function, intestinal absorption, susceptibility to coronary artery disease, thyroid and adrenal function.

The RayBiotech Total Cholesterol Colorimetric Assay Kit offers a straightforward, reliable, and highly sensitive method for measuring the concentration of total cholesterol, which encompasses both cholesteryl esters and free cholesterol, in plasma, serum, cell lysates and other biological liquid samples. This assay relies on an enzymatic reaction system that couples several essential steps. First, cholesterol esters are enzymatically hydrolyzed by cholesterol esterase, resulting in the conversion of cholesteryl esters into cholesterol and free fatty acids. Subsequently, the cholesterol, including the original content, is oxidized by cholesterol oxidase to produce cholest-4-en-3-one and hydrogen peroxide (H2O2). Finally, a Colorimetric Probe reacts with the generated hydrogen peroxide, forming a pink-colored product. The optical density measured at 500nm is directly proportional to the concentration of cholesterol within the sample.

• Microplate reader capable of measuring absorbance at 500 nm
• Precision pipettes to deliver 2 µl to 1 ml volumes
• Distilled or deionized water
• Tubes to prepare sample dilutions
• Incubator at 37°C.

Each cholesterol standard and sample should be assayed in duplicate or triplicate. A standard curve should be generated each time the assay is performed.

1. Set incubator at 37°C, and pre-heat Enzyme Mix Solution right before the assay./li>
2. Add 10 µL of the diluted cholesterol standards or samples to the 96-well microtiter plate.
3. Add 200 µL of Enzyme Mix Solution (pre-heated at 37°C) to each well.
4. Cover with the plate cover. Carefully shake the plate for a few seconds to mix.
5. Incubate the plate for 5 minutes at 37°C.
6. Immediately measure the absorbance at 500nm using a plate reader.