AAT Bioquest

Amplite® Fluorimetric Xanthine Assay Kit

Xanthine dose response was measured with Amplite® Fluorimetric Xanthine Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).
Xanthine dose response was measured with Amplite® Fluorimetric Xanthine Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).
Xanthine dose response was measured with Amplite® Fluorimetric Xanthine Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).
Ordering information
Catalog Number
Unit Size
Add to cart
Additional ordering information
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
ShippingStandard overnight for United States, inquire for international
Request quotation
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Xanthine is a purine base found in most human body tissues and fluids. A number of stimulants are derived from xanthine, including caffeine, aminophylline, IBMX, paraxanthine, pentoxifylline, theobromine, and theophylline, which can stimulate heart rate, force of contraction, cardiac arrhythmias at high concentrations. Therefore, detection of Xanthine alteration in biological samples is important for disease diagnosis and therapy monitoring. Amplite® Fluorimetric Xanthine Assay Kit provides a quick and ultrasensitive method for the measurement of xanthine. It can be performed in a convenient 96-well or 384-well microtiter-plate format. Xanthine is oxidized to uric acid in the presence of xanthine oxidase to release hydrogen peroxide, which can be specifically measured with Amplite® Red by a fluorescence microplate reader. With Amplite® Fluorimetric Xanthine Assay Kit, as low as 0.4 µM xanthine was detected in a 100 µL reaction volume.


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black


Example protocol


Protocol summary

  1. Prepare xanthine standards or test samples (50 µL)
  2. Add xanthine working solution (50 µL)
  3. Incubate at room temperature for 30 - 60 min
  4. Read fluorescence intensity at Ex/Em = 540/590 nm

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


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 substrate stock solution (250X):
Add 40 µL of DMSO (Component F) into the vial of Amplite™ Red substrate (Component A). Note: 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 assay should be performed at pH 7 - 8 (pH 7.4 is recommended) as Amplite™ Red is unstable at pH > 8.5.

2. HRP stock solution (500X):
Add 100 µL of Assay Buffer (Component B) into the vial of Horseradish Peroxidase (Component C).

3. Xanthine oxidase (XO) stock solution (100X):
Add 100 µL of Assay Buffer (Component B) into the vial of Xanthine Oxidase (Component E) to make Xanthine Oxidase (XO) stock solution (100X).


Xanthine standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13843

Add 5 µL of Xanthine Standard (Component D) into 995 µL of Assay Buffer (Component B) to get 100 µM xanthine standard solution. Take 200 µL of 100 µM xanthine standard solution to perform 1:3 serial dilutions to get serially diluted xanthine standards (X1 - X7).


Add 20 μL of Amplite™ Red Substrate stock solution (250X), 10 μL of HRP stock solution (500X), and 50 μL of Xanthine Oxidase stock solution (100X) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.08 mL. Note: Avoid direct exposure to light and use promptly.


Table 1. Layout of Xanthine standards and test samples in a black wall/solid bottom 96-well microplate. X = xanthine standard (X1 - X7, 0.137 to 100 µM); BL = blank control; TS = test sample.


Table 2. Reagent composition for each well.

X1 - X750 µLSerial Dilution (0.137 to 100 µM)
BL50 µLAssay Buffer (Component B)
TS50 µLTest Sample
  1. Prepare xanthine standards (X), blank controls (BL), and test samples (TS) according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of xanthine working solution into each well of the xanthine standards, blank control, and test samples to make the total xanthine assay volume of 100 µL/well. For a 384-well plate, add 25 µL of working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction for 30 to 60 minutes at room temperature, protected from light.

  4. Monitor the fluorescence increase with with a fluorescence plate reader at Excitation = 530 - 570 nm (optimal at 540 nm), Emission = 590 - 600 nm (optimal at 590 nm), cutoff = 570 nm.



View all 60 references: Citation Explorer
Concentration-dependent effect of sodium hypochlorite on stem cells of apical papilla survival and differentiation
Authors: Martin DE, De Almeida JF, Henry MA, Khaing ZZ, Schmidt CE, Teixeira FB, Diogenes A.
Journal: J Endod (2014): 51
Effect of hypochlorite oxidation on cholinesterase-inhibition assay of acetonitrile extracts from fruits and vegetables for monitoring traces of organophosphate pesticides
Authors: Kitamura K, Maruyama K, Hamano S, Kishi T, Kawakami T, Takahashi Y, Onodera S.
Journal: J Toxicol Sci (2014): 71
Green synthesis of carbon dots with down- and up-conversion fluorescent properties for sensitive detection of hypochlorite with a dual-readout assay
Authors: Yin B, Deng J, Peng X, Long Q, Zhao J, Lu Q, Chen Q, Li H, Tang H, Zhang Y, Yao S.
Journal: Analyst (2013): 6551
Comparative antimicrobial activities of aerosolized sodium hypochlorite, chlorine dioxide, and electrochemically activated solutions evaluated using a novel standardized assay
Authors: Thorn RM, Robinson GM, Reynolds DM.
Journal: Antimicrob Agents Chemother (2013): 2216
Analysis of the germination kinetics of individual Bacillus subtilis spores treated with hydrogen peroxide or sodium hypochlorite
Authors: Setlow B, Yu J, Li YQ, Setlow P.
Journal: Lett Appl Microbiol (2013): 259
A simple yet effective chromogenic reagent for the rapid estimation of bromate and hypochlorite in drinking water
Authors: Zhang J, Yang X.
Journal: Analyst (2013): 434
Effect of hypochlorite-based disinfectants on inactivation of murine norovirus and attempt to eliminate or prevent infection in mice by addition to drinking water
Authors: Takimoto K, Taharaguchi M, Sakai K, Takagi H, Tohya Y, Yamada YK.
Journal: Exp Anim (2013): 237
Use of pyrogallol red and pyranine as probes to evaluate antioxidant capacities towards hypochlorite
Authors: Perez-Cruz F, Cortes C, Atala E, Bohle P, Valenzuela F, Olea-Azar C, Speisky H, Aspee A, Lissi E, Lopez-Alarcon C, Bridi R.
Journal: Molecules (2013): 1638
Enhancement of anti-cholinesterase activity of aqueous samples by hypochlorite oxidation for monitoring traces of organophosphorus pesticides in water
Authors: Kanno A, Kawakami T, Takahashi Y, Onodera S.
Journal: J Toxicol Sci (2012): 389
Colorimetric determination of hypochlorite with unmodified gold nanoparticles through the oxidation of a stabilizer thiol compound
Authors: Zhang J, Wang X, Yang X.
Journal: Analyst (2012): 2806