Cholesterol is a sterol and lipid present in the cell membranes, and is transported in the bloodstream of all animals. It is used to form cell membranes and hormones, and plays important roles in cell signaling processes. Elevated levels (hypercholesterolemia) have been associated with cardiovascular diseases such as atherosclerosis; whereas, low levels (hypocholesterolemia) may be linked to depression, cancer and cerebral hemorrhage.
Simple, direct and automation-ready procedures for measuring cholesterol are very desirable. Cholesterol Assay uses a single Working Reagent that combines cholesterol ester hydrolysis, oxidation and color reaction in one step. The color intensity of the reaction product at 570nm or fluorescence intensity at lem/ex=585/530nm is directly proportional to total cholesterol concentration in the sample.
Applications:Direct Assays: cholesterol in serum, plasma, and other biological samples
Pharmacology: effects of drugs on cholesterol metabolism.
Key Features:Sensitive and accurate. Linear detection range in 96-well plate: 1 to 100mg/dL cholesterol for colorimetric assays and 0.2 to 10 mg/dL for fluorometric assays
Convenient. Room temperature assay. No 37°C heater is needed
High-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.
Kit Contents: (100 tests in 96-well plates)Assay Buffer: 20ml Enzyme Mix: 120uLDye Reagent: 120ul Standard: 1ml 300 mg/dL cholesterolStorage conditions. Store reagents at -20°C. Shelf life: 6 months after receipt. Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.
Colormetric Procedure:Important: bring all reagents to room temperature prior to assay. Serum and plasma samples should be clear and free of turbidity or precipitates. If present, precipitates should be removed by filtration or centrifugation. If not assayed immediately, samples can be stored at -20 to -80°C for at least one year
1. Standard Curve. Prepare a 10-fold diluted 100mg/dL standard (STD) by mixing 15ul 300mg/dL Standard and 435ul Assay Buffer. Further dilute standard (STD) in Assay Buffer as shown below
No STD+Assay Buffer Vol (ul) 10 x Conc. (mg/dL)1 100ul+0ul 100 1002 80ul+20ul 100 803 60ul+40ul 100 604 40ul+60ul 100 405 30ul+70ul 100 306 20ul+80ul 100 207 10ul+90ul 100 108 0ul+100ul 100 0Transfer 50ul diluted standards into wells of a clear 96-well plate
Samples: dilute samples 10-fold (e.g. 10ul sample with 90ul Assay Buffer). Transfer 50ul diluted sample in separate wells
2. For each reaction well, mix 55ul Assay Buffer with 1ul Enzyme Mix and 1ul Dye Reagent. Add 50ul of this Working Reagent to each standard and sample well. Tap plate to mix well
3. Incubate 30 min at room temperature. Read OD at 570 nm
Note: if the Sample OD is higher than the Standard OD at 100 mg/dL, dilute sample in assay buffer and repeat the assay. Multiply result by the dilution factor.
Calculation:Subtract blank OD (water, #8) from the standard OD values and plot the OD against standard concentrations. Determine the slope using linear regression fitting. The cholesterol concentration of Sample is calculated as[Cholesterol] =ODSample-ODBlankSlope(mg/dL)Note: since both the standards and samples were diluted 10-fold, no dilution factor is required.
Fluorometric Procedure:For fluorometric assays, the linear detection range is 0.2 to 10 mg/dL cholesterol. Dilute the Standards prepared in Colorimetric Procedure: 1:10 in Assay Buffer
Transfer 50ul standards and 50ul samples into separate wells of a black 96-well plate
Add 50ul Working Reagent (see Colorimetric Procedure:). Tap plate to mix
Incubate 30 min at room temperature and read fluorescence at lex=530nm and lem=585nm
If assays in 384-well plate are desired, use 5ul Standards / samples and 45ul Working Reagent
The cholesterol concentration of Sample is calculated as[Cholesterol] =FSample-FBlankSlope(mg/dL)
Materials Required, But Not Provided:Pipetting (multi-channel) devices, 96-well plate and plate reader.
