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Amplite® Fluorimetric Sphingomyelin Assay Kit *Red Fluorescence*

Sphingomyelin dose response was measured on a solid black 96-well plate with Amplite® Fluorimetric Sphingomyelin Assay Kit using a Gemini fluorescence microplate reader (Molecular Devices).
Sphingomyelin dose response was measured on a solid black 96-well plate with Amplite® Fluorimetric Sphingomyelin Assay Kit using a Gemini fluorescence microplate reader (Molecular Devices).
Sphingomyelin dose response was measured on a solid black 96-well plate with Amplite® Fluorimetric Sphingomyelin Assay Kit using a Gemini fluorescence microplate reader (Molecular Devices).
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Spectral properties
Excitation (nm)571
Emission (nm)584
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Excitation (nm)
Emission (nm)
Sphingomyelin (SM) is largely found in the exoplasmic leaflet of the cell membrane, primarily in nervous tissue. It plays an important role in signal transduction. It accumulates abnormally in Niemann-Pick disease and Abetalipoproteinemia. Our Amplite® Fluorimetric Sphingomyelin Assay Kit provides the most sensitive method for detecting neutral SM activity or screening its inhibitors. The kit uses Amplite® Red as a fluorogenic probe to indirectly quantify the phosphocholine produced from the hydrolysis of sphingomyelin (SM) by sphingomyelinase (SMase). It can be used for measuring the SM in blood, cell extracts or other solutions. The fluorescence intensity of Amplite® Red is proportional to the formation of phosphocholine, therefore to the amount of SM. Amplite® Red enables the assay readable either in fluorescence intensity or absorption mode. The kit is an optimized "mix and read" assay that can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step.


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black


Example protocol


Protocol summary

  1. Prepare SMase working solution (50 µL)
  2. Add sphingomyelin standards or test samples (50 µL)
  3. Incubate at 37 °C for 1 - 2 hours
  4. Add sphingomyelin assay mixture (50 µL)
  5. Incubate at RT for 0.5 - 2 hours
  6. Monitor fluorescence intensity at Ex/Em = 540/590 nm (cut off at 570 nm)

Important notes
Thaw kit components at room temperature before starting your 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. SMase stock solution (100X):
Add 50 µL of PBS with 0.1% BSA into the vial of Sphingomyelinase (Component B) to make SMase stock solution (100X).

2. Amplite™ Red stock solution (200X):
Add 80 µL of DMSO (Component G) into the vial of Amplite™ Red (Component C) to make 200X Amplite™ Red stock solution. Keep from light. Note: The Amplite™ Red is unstable in the presence of thiols (such as DTT and 2-mercaptoethanol). The final concentration of DTT or 2-mercaptoethanol in the reaction should be lower than 10 µM. Amplite™ Red is also unstable at high pH (>8.5). The reactions should be performed at pH 7 - 8. pH 7.4 is recommended for the assay buffer.


Sphingomyelin standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13625

Add 2 µL of 50 mM Sphingomyelin (Component F) into 1000 µL of SMase Reaction Buffer (Component D) to get a 100 µM Sphingomyelin standard solution (SM7). Take 100 µM Sphingomyelin standard solution to perform 1:3 serial dilutions to get serially diluted sphingomyelin standards (SM6 - SM1).


1. Sphingomyelinase (SMase) working solution:
Prepare SMase working solution by adding the whole content (50 µL) of 100X SMase stock solution into 5 mL of SMase Reaction Buffer (Component D) and mix well. Note: The SMase working solution should be used promptly.

2. Sphingomyelin working solution:
Add the whole content (5 mL) of Assay Buffer (Component E) into the bottle of Enzyme Mix (Component A) and mix well. Add 25 µL 200X Amplite™ Red stock solution into the bottle of Enzyme Mix solution to make the sphingomyelin assay mixture before starting the assay. Note: The sphingomyelin assay mixture should be used promptly and kept from light; longer storage is likely to cause high assay background.

For guidelines on cell sample preparation, please visit


Table 1. Layout of sphingomyelin standards and test samples in a solid black 96-well microplate. SM = Sphingomyelin Standards (SM1 - SM7, 0.1 to 100 µM), BL = Blank Control, TS = Test Samples.


Table 2. Reagent composition for each well.

SM1 - SM750 µLSerial Dilutions (0.1 to 100 µM)
BL50 µLSMase Reaction Buffer
TS50 µLtest sample
  1. Prepare sphingomyelin standards (SM), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL. Note: Treat your cells or tissue samples as desired.

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

  3. Incubate the reaction mixture at 37°C for 1 - 2 hours.

  4. Add 50 µL of sphingomyelin assay mixture to each well of sphingomyelin standard, blank control, and test samples to make the total sphingomyelin assay volume of 150 µL/well. For a 384-well plate, add 25 µL of sphingomyelin assay mixture into each well instead, for a total volume of 75 µL/well.

  5. Incubate the reaction mixture for 1 - 2 hours at room temperature (protected from light).

  6. Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 540/590 nm (cut off at 570 nm).


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Spectral properties

Excitation (nm)571
Emission (nm)584



View all 9 citations: Citation Explorer
Sphingomyelin synthase 1 supports two steps of rubella virus life cycle
Authors: Yagi, Mayuko and Hama, Minami and Ichii, Sayaka and Nakashima, Yurie and Kanbayashi, Daiki and Kurata, Takako and Yusa, Kosuke and Komano, Jun
Journal: iScience (2023)
Polyphyllin D “Punctures” Hypertrophic Lysosomes to Reverse Drug Resistance of Hepatocellular Carcinoma by Targeting Acid Sphingomyelinase
Authors: Wang, Yang and Chen, Yan-Yan and Gao, Gui-Bin and Zheng, Yang-Han and Yu, Nan-Nan and Ouyang, Lan and Gao, Xuejuan and Li, Nan and Wen, Shi-Yuan and Huang, Shangjia and others,
Journal: Molecular Therapy (2023)
Sphingomyelin synthase activity affects TRIF-dependent signaling of Toll-like receptor 4 in cells stimulated with lipopolysaccharide
Authors: Prymas, Kamila and Swiatkowska, Anna and Traczyk, Gabriela and Ziemlińska, Ewelina and Dziewulska, Anna and Ciesielska, Anna and Kwiatkowska, Katarzyna
Journal: Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids (2019): 158549
Doxepin mitigates noise induced neuronal damage in primary auditory cortex of mice via suppression of acid sphingomyelinase/ceramide pathway
Authors: Su, Yu-Ting and Meng, Xing-Xing and Zhang, Xi and Guo, Yi-Bin and Zhang, Hai-Jun and Cheng, Yao-Ping and Xie, Xiao-Ping and Chang, Yao-Ming and Bao, Jun-Xiang
Journal: The Anatomical Record (2017)
Riccardin DN induces lysosomal membrane permeabilization by inhibiting acid sphingomyelinase and interfering with sphingomyelin metabolism in vivo
Authors: Li, Lin and Niu, Huanmin and Sun, Bin and Xiao, Yanan and Li, Wei and Yuan, Huiqing and Lou, Hongxiang
Journal: Toxicology and Applied Pharmacology (2016): 175--184
New Aspects of Silibinin Stereoisomers and their 3-O-galloyl Derivatives on Cytotoxicity and Ceramide Metabolism in Hep G2 hepatocarcinoma Cell Line
Authors: Mashhadi Akbar Boojar, Mahdi and Ejtemaei Mehr, Shahram and Hassanipour, Mahsa and Mashhadi Akbar Boojar, Masoud and Dehpour, Ahmad Reza
Journal: Iranian Journal of Pharmaceutical Research (2016): 421--433
Mitochondrial respiration controls lysosomal function during inflammatory T cell responses
Authors: Baixauli, Francesc and Acín-Pérez, Rebeca and Villarroya-Beltrí, Carolina and Mazzeo, Carla and Nunez-Andrade, Norman and Gab, undefined and é-Rodriguez, Enrique and Ledesma, Maria Dolores and Blázquez, Alberto and Martin, Miguel Angel and Falcón-Pérez, Juan Manuel and others, undefined
Journal: Cell metabolism (2015): 485--498
The ATP-binding cassette transporter-2 (ABCA2) regulates esterification of plasma membrane cholesterol by modulation of sphingolipid metabolism
Authors: Davis, Warren
Journal: Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids (2014): 168--179
A high-throughput sphingomyelinase assay using natural substrate
Authors: Xu, Miao and Liu, Ke and Southall, Noel and Marugan, Juan J and Remaley, Alan T and Zheng, Wei
Journal: Analytical and bioanalytical chemistry (2012): 407--414


View all 65 references: Citation Explorer
Expression of alkaline sphingomyelinase in yeast cells and anti-inflammatory effects of the expressed enzyme in a rat colitis model
Authors: Andersson D, Kotarsky K, Wu J, Agace W, Duan RD.
Journal: Dig Dis Sci (2009): 1440
A novel sphingomyelinase-like enzyme in Ixodes scapularis tick saliva drives host CD4 T cells to express IL-4
Authors: Alarcon-Chaidez FJ, Boppana VD, Hagymasi AT, Adler AJ, Wikel SK.
Journal: Parasite Immunol (2009): 210
A novel mitochondrial sphingomyelinase in zebrafish cells
Authors: Yabu T, Shimuzu A, Yamashita M.
Journal: J Biol Chem (2009): 20349
Alterations of myelin-specific proteins and sphingolipids characterize the brains of acid sphingomyelinase-deficient mice, an animal model of Niemann-Pick disease type A
Authors: Buccinna B, Piccinini M, Prinetti A, Sc and roglio F, Prioni S, Valsecchi M, Votta B, Grifoni S, Lupino E, Ramondetti C, Schuchman EH, Giordana MT, Sonnino S, Rinaudo MT.
Journal: J Neurochem (2009): 105
A complex extracellular sphingomyelinase of Pseudomonas aeruginosa inhibits angiogenesis by selective cytotoxicity to endothelial cells
Authors: Vasil ML, Stonehouse MJ, Vasil AI, Wadsworth SJ, Goldfine H, Bolcome RE, 3rd, Chan J.
Journal: PLoS Pathog (2009): e1000420
Structure and function of sphingomyelinase
Authors: Oda M., undefined
Journal: Yakugaku Zasshi (2009): 1233
ApoCIII-enriched LDL in type 2 diabetes displays altered lipid composition, increased susceptibility for sphingomyelinase, and increased binding to biglycan
Authors: Hiukka A, Stahlman M, Pettersson C, Levin M, Adiels M, Teneberg S, Leinonen ES, Hulten LM, Wiklund O, Oresic M, Olofsson SO, Taskinen MR, Ekroos K, Boren J.
Journal: Diabetes (2009): 2018
Purification, characterization, and gene cloning of sphingomyelinase C from Streptomyces griseocarneus NBRC13471
Authors: Sugimori D., undefined
Journal: J Biosci Bioeng (2009): 293
Transcriptional regulation of neutral sphingomyelinase 2 gene expression of a human breast cancer cell line, MCF-7, induced by the anti-cancer drug, daunorubicin
Authors: Ito H, Murakami M, Furuhata A, Gao S, Yoshida K, Sobue S, Hagiwara K, Takagi A, Kojima T, Suzuki M, Banno Y, Tanaka K, Tamiya-Koizumi K, Kyogashima M, Nozawa Y, Murate T.
Journal: Biochim Biophys Acta (2009): 681
Neutral sphingomyelinase-induced ceramide accumulation by oxidative stress during carbon tetrachloride intoxication
Authors: Ichi I, Kamikawa C, Nakagawa T, Kobayashi K, Kataoka R, Nagata E, Kitamura Y, Nakazaki C, Matsura T, Kojo S.
Journal: Toxicology (2009): 33