Amplite® Cholesterol Quantitation Kit

Ordering information

Price
Catalog Number
Unit Size
Quantity

Add to cart

Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

Spectral properties

Excitation (nm)	571
Emission (nm)	584

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Overview SDS Protocol

Excitation (nm)

571

Emission (nm)

584

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.

Platform

Fluorescence microplate reader

Excitation	540 nm
Emission	590 nm
Cutoff	570 nm
Recommended plate	Solid black

Components

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

Spectral properties

Excitation (nm)	571
Emission (nm)	584

Product Family

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

Images

Figure 1. Cholesterol dose response was measured with Amplite® Fluorimetric Cholesterol Quantitation Kit in a black 96-well plate using a Gemini fluorescence microplate reader (molecular devices). As low as 0.03 µM cholesterol can be detected with 30 minutes incubation (n=3).

Figure 2. Inhibition of cholesterol and sphingomyelin affected RABV budding and release. (A) Effect of MβCD on BSR cell viability. BSR cells seeded in 96-well microplates were untreated or pre-treated with 0.5–8 mM MβCD for 1 h. The supernatants were removed and washed twice with PBS. An MTT assay was then performed. (B) Effect of MβCD on cholesterol and RABV replication. Cells seeded in six-well microplates were infected with rRC-HL at an MOI of 0.001 and then treated with 0.5–8 mM MβCD for 24 h at 37 °C. The supernatants were harvested for viral titration. The cells were assayed for cholesterol content using a cholesterol quantitation kit (40006; AAT Bioquest, Inc.) according to the manufacturer’s specfications. (C) Effect of MβCD on RABV replication. Based on A and B, a set of the cells was used to prepare cell lysates that were subjected to Western blot analysis for RABV N and β-actin protein expression. (D) The N protein/actin ratios in Fig. 3C were measured using Li-Cor Odyssey 3.0 analytical software version 29. The error bars were calculated from at least 3 independent inhibition tests. (E) Effect of myriocin on BSR cell viability. BSR cells were seeded in 96-well microplates and either untreated or pre-treated with 0.01–100 μm myriocin for 1 h. The supernatants were removed and washed twice with PBS. An MTT assay was then performed. (F) Effects of myriocin on sphingomyelin content and RABV replication. Cells seeded in six-well microplates were infected with rRC-HL at an MOI of 0.001 and then treated with 0.5–50 μm myriocin for 24 h at 37 °C. The supernatants were harvested for viral titration. The cells were assayed for sphingomyelin content using a sphingomyelin colorimetric assay kit (10009928). (G) Effect of myriocin on RABV replication. Based on D and E, another set of cells was used to prepare lysates that were then subjected to Western blotting analysis for RABV N and β-actin protein expression. (H) The N protein/actin ratios in Fig. 3G were measured using Li-Cor Odyssey 3.0 analytical software version 29. Source: Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4 by Tang et al., Scientific Reports, July 2016.

Citations

View all 13 citations: Citation Explorer

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)

Viperin impairs the innate immune response through the IRAK1-TRAF6-TAK1 axis to promote Mtb infection
Authors: Zhou, Xinying and Zhang, Zelin and Xu, Hui and Zhu, Bo and Zhang, Lijie and Lie, Linmiao and Huang, Yingqi and Du, Xialin and Liu, Honglin and Li, Yanfen and others,
Journal: Science Signaling (2022): eabe1621

Inhibition of human sperm function by an antibody against apolipoprotein A1: A protein located in human spermatozoa
Authors: Chi, Xiuping and Xiang, Daijun and Sha, Yingjiao and Liang, Shuang and Wang, Chengbin
Journal: Andrologia (2022): e14365

Viperin deficiency promotes dendritic cell activation and function via NF-kappaB activation during Mycobacterium tuberculosis infection
Authors: Zhou, Xinying and Xu, Hui and Li, Qianna and Wang, Qi and Liu, Honglin and Huang, Yingqi and Liang, Yao and Lie, Linmiao and Han, Zhenyu and Chen, Yaoxin and others,
Journal: Inflammation Research (2022): 1--15

In vitro characterization revealed five new potential pathogenic variants in the APOB gene
Authors: Di Taranto, MD and Galicia, U and Larrea, A and Giacobbe, C and Calcaterra, I and Palma, D and Cardiero, G and Iannuzzo, G and Di Minno, MND and Iannuzzi, A and others,
Journal: Atherosclerosis (2021): e183--e184

A correlation between DLCN score and serum creatinine in patients with familial hypercholesterolaemia (FH) in Lithuania
Authors: Skiauteryte, E and Gabartaite, D and Aliosaitiene, U and Petrulioniene, Z and Rinkuniene, E and Dzenkeviciute, V and Petrulionyte, E and Barysiene, J and Cerkauskiene, R
Journal: Atherosclerosis (2021): e183

Performance assessment of salivary screening in familial hypercholesterolemia in children
Authors: Fricaudet, M and Di-Filippo, M and Moulin, P and Poinsot, P and Sage, C and Brignot, H{\'e}l{\`e}ne and Feron, Gilles and Charri{\`e}re, S and Peretti, N
Journal: Atherosclerosis (2021): e183

The interferon stimulated gene viperin, restricts Shigella. flexneri in vitro
Authors: Helbig, KJ and Teh, MY and Crosse, KM and Monson, EA and Smith, M and Tran, EN and St, undefined and ish, AJ and Morona, R and Beard, MR
Journal: Scientific Reports (2019): 1--12

Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4
Authors: Tang, Hai-Bo and Lu, Zhuan-Ling and Wei, Xian-Kai and Zhong, Tao-Zhen and Zhong, Yi-Zhi and Ouyang, Ling-Xuan and Luo, Yang and Xing, Xing-Wei and Liao, Fang and Peng, Ke-Ke and others, undefined
Journal: Scientific Reports (2016)

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

Genetic-epidemiological evidence on genes associated with HDL cholesterol levels: a systematic in-depth review
Authors: Boes E, Coassin S, Kollerits B, Heid IM, Kronenberg F.
Journal: Exp Gerontol (2009): 136

Systematic review, meta-analysis and regression of randomised controlled trials reporting an association between an intake of circa 25 g soya protein per day and blood cholesterol
Authors: Harl, undefined and JI, Haffner TA.
Journal: Atherosclerosis (2008): 13

Does initial breastfeeding lead to lower blood cholesterol in adult life? A quantitative review of the evidence
Authors: Owen CG, Whincup PH, Kaye SJ, Martin RM, Davey Smith G, Cook DG, Bergstrom E, Black S, Wadsworth ME, Fall CH, Freudenheim JL, Nie J, Huxley RR, Kolacek S, Leeson CP, Pearce MS, Raitakari OT, Lisinen I, Viikari JS, Ravelli AC, Rudnicka AR, Strachan DP, Williams SM.
Journal: Am J Clin Nutr (2008): 305

High-density lipoprotein-cholesterol and risk of stroke and carotid atherosclerosis: a systematic review
Authors: Amarenco P, Labreuche J, Touboul PJ.
Journal: Atherosclerosis (2008): 489

Effect of HMG CoA reductase inhibitors on low-density lipoprotein cholesterol and C-reactive protein: systematic review and meta-analysis
Authors: Genser B, Grammer TB, Stojakovic T, Siekmeier R, Marz W.
Journal: Int J Clin Pharmacol Ther (2008): 497