Amplite® Cholesterol Quantitation Kit

Cholesterol is required to build and maintain cell membranes. It modulates membrane fluidity over the range of physiological temperatures. Within cells, cholesterol is the precursor molecule in several biochemical pathways. Cholesterol is also an important precursor molecule for the synthesis of Vitamin D and the steroid hormones, including the adrenal gland hormones cortisol and aldosterone as well as the sex hormones progesterone, estrogens, together with testosterone and their derivatives. This Amplite® Cholesterol Quantitation Assay Kit provides one of the most sensitive methods for quantifying cholesterol. The kit uses Amplite® Red to quantify the concentration of cholesterol, which is related to the production of hydrogen peroxide in the cholesterol oxidase-mediated enzyme coupling reactions in the presence of cholesterol. The amount of cholesterol is proportional to the concentration of hydrogen peroxide formed in the enzyme coupling reaction cycle. In the presence of peroxidase, the fluorescence intensity of Amplite® Red is proportional to the concentration of hydrogen peroxide that is converted to the concentration of cholesterol. The assay can be readily read with a fluorescence microplate reader.

Example protocol

AT A GLANCE

Protocol Summary

Prepare Cholesterol Assay working solution (50 µL)
Add cholesterol standards and/or test samples (50 µL)
Incubate at 37°C for 30 minutes
Monitor fluroscence intensity at Ex/Em = 540/590 nm

Important Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Amplite™ Red stock solution (250X)

Add 40 µL of DMSO (Component E) into the vial of Amplite™ Red substrate (Component A). The stock solution should be used promptly. Any remaining solution should be aliquoted and refrozen at -20 ^oC.
Note Avoid repeated freeze-thaw cycles.
Note The Amplite™ Red substrate is unstable in the presence of thiols such as dithiothreitol (DTT) and 2-mercaptoethanol. The final concentration of DTT or 2-mercaptoethanol in the reaction should be no higher than 10 µM. The Amplite™ Red substrate is also unstable at high pH (> 8.5). Therefore, the reaction should be performed at pH 7–8. The provided assay buffer (pH 7.4) is recommended.

2. Cholesterol standard stock solution (20 µM)

Add 10 µL of Cholesterol Standard (Component D) into 990 µL of Assay Buffer (Component B) and mix well.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/40006

Cholesterol standard

Prepare a cholesterol standard (20 µM). Then perform 1:3 serial dilutions in Assay Buffer (Component B) to get approximately 10, 3, 1, 0.3, 0.1, 0.03 and 0.01 µM serially diluted cholesterol standards. A non-cholesterol buffer control is included as blank control.

PREPARATION OF WORKING SOLUTION

Cholesterol Assay working solution

Add 5 mL of Assay Buffer (Component B) into the bottle of Cholesterol Enzyme Mix (Component C), and mix them well. Add 20 µL of Amplite Red™ stock solution (250X) into the Cholesterol Enzyme Mix bottle.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of Cholesterol standards and test samples in a solid black 96-well microplate. CS = Cholesterol standard (CS1-CS7); BL = blank control; TS = test sample.

BL	BL	TS	TS
CS1	CS1	...	...
CS2	CS2	...	...
CS3	CS3
CS4	CS4
CS5	CS5
CS6	CS6
CS7	CS7

Table 2. Reagent composition for each well

well	Volume	Reagent
CS1 - CS7	50 µL	Serial Dilutions (0.01 to 10 µM)
BL	50 µL	Assay Buffer (Component B)
TS	50 µL	test sample

Cholesterol assay

Add cholesterol standards and cholsterol containing test samples into a 96-well solid black microplate as described in Tables 1 and 2.
Add 50 µL of Cholesterol Assay working solution into each well of cholesterol standard, blank control, and test samples (Table 2) to make the total cholesterol assay volume of 100 µL/well. Note: For a 384-well plate, add 25 µL of sample and 25 µL of assay reaction mixture into each well.
Incubate the reaction for 30 minutes at 37 ^oC, protected from light.
Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em= 530-570 nm/590-600 nm (optimal Ex/Em = 540/590 nm). Note: The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 576±5 nm. The absorption detection has lower sensitivity compared to the fluorescence reading.

Spectrum

Open in Advanced Spectrum Viewer

Product family

Name	Excitation (nm)	Emission (nm)
Amplite® Choline Quantitation Kit	571	584
Amplite® Ethanol Quantitation Kit	571	584
Amplite® Lactose Quantitation Kit	571	584

Citations

View all 16 citations: Citation Explorer

Cholesterol depletion suppresses thermal necrosis resistance by alleviating an increase in membrane fluidity
Authors: Kanamori, Taisei and Yasuda, Shogo and Duan, Runjing and Ohashi, Mei and Amou, Mai and Hori, Kanato and Tsuda, Ryota and Fujimoto, Taiki and Higashi, Kenjirou and Xu, Wei and others,
Journal: Scientific Reports (2025): 10133

OBP2A regulates epidermal barrier function and protects against cytotoxic small hydrophobic molecules
Authors: Nakanishi, Shinobu and Hasegawa, Tatsuya and Maeno, Katsuyuki and Motoyama, Akira and Denda, Mitsuhiro
Journal: iScience (2024)

M$\beta$CD inhibits SFTSV entry by disrupting lipid raft structure of the host cells
Authors: Cheng, Min and Zhang, Rui and Li, Jianshu and Ma, Wenyuan and Li, Linrun and Jiang, Na and Liu, Bingxin and Wu, Jing and Zheng, Nan and Wu, Zhiwei
Journal: Antiviral Research (2024): 106004

Redefine the role of D-$\alpha$-Tocopheryl polyethylene glycol 1000 succinate on P-glycoprotein, multidrug resistance protein 1, and breast cancer resistance protein mediated cancer multidrug resistance
Authors: Chen, Jing-Yi and Sung, Chieh-Ju and Chen, Ssu-Chi and Hsiang, Yi-Ping and Hsu, Yung-Chia and Teng, Yu-Ning
Journal: European Journal of Pharmaceutical Sciences (2023): 106579

Salivary cholesterol level does not reflect cholesterolemia in children with heterozygous familial hypercholesterolemia
Authors: Fricaudet, Marianne and Di Filippo, Mathilde and Moulin, Philippe and Nony, S{\'e}verine and Peron, Marie Anais and Brignot, H{\'e}l{\`e}ne and Feron, Gilles and Sage, C{\'e}dric and Poinsot, Pierre and Loras, R{\'e}mi Duclaux and others,
Journal: Nutrition Clinique et M{\'e}tabolisme (2023)

References

View all 101 references: Citation Explorer

Lowering cholesterol - a review on the role of plant sterols
Authors: Clifton P., undefined
Journal: Aust Fam Physician (2009): 218

Prospective studies on the relationship between high-density lipoprotein cholesterol and cardiovascular risk: a systematic review
Authors: Chirovsky DR, Fedirko V, Cui Y, Sazonov V, Barter P.
Journal: Eur J Cardiovasc Prev Rehabil (2009): 404

Systematic review and metaanalysis of statins for heterozygous familial hypercholesterolemia in children: evaluation of cholesterol changes and side effects
Authors: O'Gorman CS, Higgins MF, O'Neill MB.
Journal: Pediatr Cardiol (2009): 482

Ezetimibe monotherapy for cholesterol lowering in 2,722 people: systematic review and meta-analysis of randomized controlled trials
Authors: P, undefined and or A, Ara RM, Tumur I, Wilkinson AJ, Paisley S, Duenas A, Durrington PN, Chilcott J.
Journal: J Intern Med (2009): 568

Association between change in high density lipoprotein cholesterol and cardiovascular disease morbidity and mortality: systematic review and meta-regression analysis
Authors: Briel M, Ferreira-Gonzalez I, You JJ, Karanicolas PJ, Akl EA, Wu P, Blechacz B, Bassler D, Wei X, Sharman A, Whitt I, Alves da Silva S, Khalid Z, Nordmann AJ, Zhou Q, Walter SD, Vale N, Bhatnagar N, O'Regan C, Mills EJ, Bucher HC, Montori VM, Guyatt GH.
Journal: Bmj (2009): b92

Page updated on September 3, 2025

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