BioReagent BioReagent for sensitive chromatographic and analytical workflows requiring minimal baseline interference.
Protected from light,Store at -20°C Ships Ice chest + Ice pads Check lot-specific COA for exact specifications.
SDS, COA, datasheet, and spec sheet available for download. Lot-specific COA accessible via lot number lookup.
Cited in 0 peer-reviewed publications across chromatography, organic synthesis, and cross-coupling reactions.
Free Cholesterol (FC) is the main component of cell membranes and an important precursor for the synthesis of physiological active substances such as adrenal corticosteroids, sex hormones, bile acids, and vitamin D. Tissue FC concentration can serve as an indicator of lipid metabolism.
Detection Principle: Cholesterol oxidase catalyzes the conversion of FC to Δ4-cholestenone and hydrogen peroxide. Peroxidase then catalyzes the reaction of hydrogen peroxide, 4-aminoantipyrine, and phenol to produce a red quinoneimine compound, which has an absorption peak at 500 nm. The color intensity is proportional to the FC content.
Detection Range: 0.09 - 9 µmoL/mL
Sensitivity: 0.09 µmoL/mL
Applicable Samples: Serum (plasma), animal tissues, cells, bacteria
| Component | 48T | 96T | Storage |
| Reagent Ⅰ | 1EA | 1EA | -20℃. Store in the dark. |
| Reagent Ⅱ | 5 mL | 10 mL | 2-8℃ |
| Reagent Ⅲ | 10 mL | 20 mL | 2-8℃. Store in the dark. |
| Standard | 1 mL | 1 mL | 2-8℃. Store in the dark. |
Note: Before formal testing, it is recommended to perform a preliminary test with 2-3 samples expected to have significant differences.
User-Prepared Instruments and Reagents
Microplate reader or visible spectrophotometer (capable of measuring absorbance at 505 nm)
96-well plates and micro glass cuvettes, adjustable micropipettes and tips
Refrigerated centrifuge, ice maker, water bath
Anhydrous ethanol
Homogenizer (for tissue samples)
Experimental Procedure
1. Reagent Preparation
| Reagent Name | Reagent Preparation | Notes |
| Working ReagentⅠ | Prepare before use: For 48T, add 4 mL Reagent II; for 96T, add 8 mL Reagent II. Dissolve completely. | Unused reagent can be stored at 4°C for one week or aliquoted and stored at -20°C protected from light for one month. Avoid repeated freeze-thaw cycles. |
| Reagent Ⅱ | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| Reagent Ⅲ | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C protected from light. |
| Working Solution | Prepare immediately before use: For each well, prepare 200 μL by mixing Working Reagent I and Reagent III in a 1:3 ratio. Mix well. | |
| Standard | Ready-to-use; equilibrate to room temperature before use. | Store at -20°C protected from light. |
2. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for one month.
2.1 Animal Tissue
Weigh approximately 0.1 g of tissue. Add 1 mL of anhydrous ethanol and homogenize in an ice bath. Centrifuge at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.
2.2 Cells, Bacteria
Collect 5 million cells or bacteria into a centrifuge tube; discard the supernatant. Add 1 mL of anhydrous ethanol. Disrupt the cells/bacteria by sonication for 5 minutes (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Centrifuge at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.
2.3 Serum (Plasma)
Assay directly.
Note: The extraction buffer contains components that denature proteins. If calculating based on protein concentration, protein needs to be re-extracted separately using deionized water. If protein concentration measurement is required, Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.
3. Assay Steps
3.1 Preheat the microplate reader or visible spectrophotometer for 30 minutes. Set the wavelength to 500 nm. For spectrophotometers, zero the instrument with deionized water.
3.2 Assay Procedure (perform in a 96-well plate or micro glass cuvette):
| Reagent | Blank Well (μL) | Standard Well (μL) | Test Well (μL) |
| Anhydrous Ethanol | 5 | 0 | 0 |
| Standard | 0 | 5 | 0 |
| Sample | 0 | 0 | 5 |
| Working Solution | 200 | 200 | 200 |
Mix well and incubate at 37°C for 30 minutes. Measure the absorbance at 505 nm, recorded as Ablank, Astandard, and Atest. Calculate ΔAtest = Atest - Ablank and ΔAstandard = Astandard - Ablank.
Note: The Blank and Standard wells only need to be set up once. A preliminary test with 2-3 samples showing expected significant differences is recommended. If ΔAtest is less than 0.01, consider increasing the sample volume appropriately. If ΔAtest is greater than 1.0, further dilute the sample with anhydrous ethanol (multiply the result by the dilution factor) or reduce the sample amount used for extraction. The detection time should not exceed 1 hour.
4. Calculation of Results
Note: We provide both the derived formula and a simplified formula. They are equivalent. It is recommended to use the simplified formula in bold for final calculation.
4.1 Sample FC Content Calculation
(1) Based on Sample Mass
Derived Formula:
FC Content (μmol/g fresh weight) = CStandard × ΔAtest ÷ ΔAstandard × Vsample ÷ (W × Vsample ÷ Vtotal sample)
Simplified Formula:
FC Content (μmol/g fresh weight) = 1.25 × ΔAtest ÷ ΔAstandard ÷ W
(2) Based on Protein Concentration
Derived Formula:
FC Content (μmol/mg prot) = CStandard × V × ΔAtest ÷ ΔAstandard × Vsample ÷ (Cpr × Vsample)
Simplified Formula:
FC Content (μmol/mg prot) = 5 × ΔAtest ÷ ΔAstandard ÷ Cpr
(3) Based on Bacterial or Cell Count
Derived Formula:
FC Content (μmol/10⁴) = CStandard × ΔAtest ÷ ΔAstandard × Vsample ÷ (500 × Vsample ÷ Vtotal sample)
Simplified Formula:
FC Content (μmol/10⁴) = 0.01 × ΔAtest ÷ ΔAstandard
(4) Based on Liquid Volume
Derived Formula:
FC Content (μmol/mL) = CStandard × ΔAtest ÷ ΔAstandard
Simplified Formula:
FC Content (μmol/mL) = 5 × ΔAtest ÷ ΔAstandard
Parameter Definitions:
CStandard: 5 mmol/L = 5 μmol/mL
Cpr: Sample protein concentration (mg/mL)
Vsample: Volume of sample added to the reaction system (0.005 mL)
W: Sample fresh weight (g)
Vtotal sample: Volume of extraction buffer added during sample preparation (1 mL)
500: Total number of cells or bacteria (5 million)
5. Representative Results
Taking 5 μL of mouse serum and following the assay steps:
Measured: Atest = 0.178, Ablank = 0.0048, Astandard = 0.582
Calculated: ΔAtest = 0.178 - 0.048 = 0.13, ΔAstandard = 0.582 - 0.048 = 0.534
Calculated FC Content (μmol/mL) = 5 × ΔAtest ÷ ΔAstandard = 5 × 0.13 ÷ 0.534 = 1.22 μmol/mL
Precautions
1. Before formal testing, it is recommended to perform a preliminary test with 2-3 samples expected to have significant differences.
2. This product is for research use only. Not for use in clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.
Comprehensive hazard, handling, storage, and regulatory compliance document.
Download SDS →Lot-specific quality data. Enter your lot number to retrieve the exact COA.
Look up COA →Full quality attributes and acceptance criteria for this grade.
View spec sheet →Our grade selection guide covers purity, stabilizer status, and application suitability for all variants in our catalog.
View BioReagent grade guide →
