Amplite® Fluorimetric Acidic Sphingomyelinase Assay Kit Red Fluorescence

Name: Amplite® Fluorimetric Acidic Sphingomyelinase Assay Kit *Red Fluorescence*
Brand: AAT Bioquest
SKU: 13622
Price: 341 USD
Availability: InStock

Sphingomyelinase (SMase) is an enzyme that is responsible for cleaving sphingomyelin (SM) to phosphocholine and ceramide. Activation of SMases plays an important role in the cellular response such as regulation of cell growth, cell differentiation, cell cycle arrest and programmed cell death. Five types of sphingomyelinase (SMase) have been identified based on their cation dependence and pH optima of action, including lysosomal acid SMase, secreted zinc-dependent acid SMase, magnesium-dependent neutral SMase, magnesium-independent neutral SMase and alkaline SMase. Among them, the lysosomal acidic SMase and the magnesium-dependent neutral SMase are considered to be the major factors for the production of ceramide in cellular stress responses. Our Amplite® Fluorimetric Acidic Sphingomyelinase Assay Kit provides one of the most sensitive methods for detecting acidic SMase 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 SMase activity in blood, cell extracts or other solutions. The fluorescence intensity of Amplite® Red is proportional to the formation of phosphocholine, therefore to the SMase activity. The kit is an optimized "mix and read" assay compatible with HTS liquid handling instruments.

Example protocol

AT A GLANCE

Protocol Summary

Prepare acidic SMase standards or SMase test samples (50 µL)
Add sphingomyelin working solution (50 µL)
Incubate at 37 °C for 2 - 3 hours
Add sphingomyelinase working solution (50 µL)
Incubate at RT for 1 - 2 hours
Monitor fluorescence increase at Ex/Em = 540/590 nm (cut off at 570 nm)

Important Thaw 1 vial (or bottle) of each kit component to room temperature before starting your 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.

Amplite™ Red stock solution (200X)

Add 80 µL of DMSO (Component F) into the vial of Amplite™ Red (Component C) to make 200X Amplite™ Red stock solution.
Note 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. The assay buffer at pH 7.4 is recommended.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/13622

Acidic sphingomyelinase standard

Dilute acidic sphingomyelinase stock solution in 20 mM sodium acetate buffer (pH = 5.0, not provided in the kit). We recommend the concentration range from 10 U/mL to 0.5 U/mL. Note: Acidic sphingomyelinase standard (from human placenta) can be obtained from Sigma-Aldrich (S-5383). Diluted acidic sphingomyelinase standard solution is unstable, and should be used within 4 hours.

PREPARATION OF WORKING SOLUTION

1. Sphingomyelin working solution

Add 50 µL of Sphingomyelin (Component B) to 5 mL of SMase Reaction Buffer (Component D) and mix well.
Note The sphingomyelin working solution should be used promptly.

2. Sphingomyelinase working solution

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

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of acidic sphingomyelinase standards and test samples in a solid black 96-well microplate. SMase = Acidic Sphingomyelinase Standards (SMase1-SMase7, 0.5 to 10 U/mL); BL = Blank Control; TS = Test Samples.

BL	BL	TS	TS
SMase1	SMase1	...	...
SMase2	SMase2	...	...
SMase3	SMase3
SMase4	SMase4
SMase5	SMase5
SMase6	SMase6
SMase7	SMase7

Table 2. Reagent composition for each well

Well	Volume	Reagent
SMase1 - SMase7	50 µL	Serial Dilution (0.5 to 10 U/mL)
BL	50 µL	20 mM Sodium Acetate Buffer (pH = 5)
TS	50 µL	Test Sample

Add the acidic sphingomyelinase standards and sphingomyelinase-containing test samples into a solid black 96-well microplate as shown 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.
Add 50 µL of sphingomyelin working solution into each well of the sphingomyelinase standards, blank control and test samples. Add the diluted acidic sphingomyelinase standards in duplicate. For a 384-well plate, add 25 µL of sphingomyelin working solution into each well instead, for a total volume of 50 µL/well.
Incubate the reaction mixture at 37 °C for 2 - 3 hours.
Add 50 µL of sphingomyelinase working solution into each well of the acidic sphingomyelinase standards, blank control, and test samples to make the total sphingomyelinase assay volume of 150 µL/well. For a 384-well plate, add 25 µL sphingomyelinase assay working solution into each well instead, for the total sphingomyelin assay volume of 75 µL/well.
Incubate the enzyme reaction mixture for 1 - 2 hours at room temperature (protected from light).
Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 540/590 nm (cut off at 570 nm).

Spectrum

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Citations

View all 9 citations: Citation Explorer

Extracellular high molecular weight $\alpha$-synuclein oligomers induce cell death by disrupting the plasma membrane
Authors: Ito, Naohito and Tsuji, Mayumi and Adachi, Naoki and Nakamura, Shiro and Sarkar, Avijite Kumer and Ikenaka, Kensuke and Aguirre, C{\'e}sar and Kimura, Atsushi Michael and Kiuchi, Yuji and Mochizuki, Hideki and others,
Journal: npj Parkinson's Disease (2023): 139

Sortilin deletion in the prefrontal cortex and hippocampus ameliorates depressive-like behaviors in mice via regulating ASM/ceramide signaling
Authors: Chen, Shu-jian and Gao, Cong-cong and Lv, Qun-yu and Zhao, Meng-qi and Qin, Xiao-ying and Liao, Hong
Journal: Acta Pharmacologica Sinica (2021): 1--15

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): 2220--2232

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

References

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

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