Sphingomyelinase assay kit

Manufactured by Abcam

Sourced in United Kingdom

The Sphingomyelinase Assay kit is a laboratory tool designed to measure the activity of the enzyme sphingomyelinase. Sphingomyelinase plays a role in the breakdown of sphingomyelin, a component of cell membranes. The assay kit provides a quantitative method to assess sphingomyelinase activity in biological samples.

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4 protocols using sphingomyelinase assay kit

Multiparametric Cellular Assay Protocol

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Acridine orange, 2,3-diaminonaphthalene (DAN), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazol-carbocyanine iodide (JC-1), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH₂-DA), and Autophagy Assay kit were obtained from Sigma Aldrich cat. no.:MAK138 (St. Louis, Missouri, USA). Human MAP1LC3A (Microtubule-associated proteins1A/1B light chain 3A) ELISA Kit was obtained from Fine-test (Wuhan, Hubei, China). The Fluo-4 NW Calcium Assay Kit was obtained from Molecular Probes (Eugene, USA). The Sphingomyelinase Assay kit was purchased from Abcam (Cambridge, UK). Culture media (DMEM, RPMI 1640) and fetal bovine serum (FBS) were obtained from Cambrex (Basel, Switzerland); trypsin-EDTA, penicillin and streptomycin were acquired from Sigma Aldrich (St. Louis, Missouri, USA).

Rogalska A., Gajek A., Łukawska M., Oszczapowicz I, & Marczak A. (2018). Novel oxazolinoanthracyclines as tumor cell growth inhibitors—Contribution of autophagy and apoptosis in solid tumor cells death. PLoS ONE, 13(7), e0201296.

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Quantifying Neutral Sphingomyelinase Activity

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Neutral-SMase activity was measured in cell extracts via a sphingomyelinase assay kit (Abcam, Catalog # ab138876, Cambridge, UK). This assay utilizes sphingomyelin as substrate to specifically monitor SMase activity. First, SMase hydrolyses sphingomyelin to yield ceramide and phosphocholine. The absorbance of the colorimetric probe at 655 nm is proportional to the formation of phosphocholine, therefore to the SMase activity. A standard curve of absorbance values of known amounts of sphingomyelinase standards was generated. Sphingomyelinase activity in the samples (mU/ml) were calculated from their corresponding absorbance values via the standard curve.

Afşar E., Kırımlıoglu E., Çeker T., Yılmaz Ç., Demir N, & Aslan M. (2020). Effect of ER stress on sphingolipid levels and apoptotic pathways in retinal pigment epithelial cells. Redox Biology, 30, 101430.

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Quantitative Assay of nSMase Activity

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Duplicate aliquots of 5 μg surface membrane extract proteins of untreated and ARA (50 μM, 30 min)-treated cells were evaluated for nSMase content using the Sphingomyelinase Assay kit of Abcam, ab287874 following the manufacturer’s instruction. Fluorescence was measured at Ex/Em of 540/590 nm, at 30, 60, and 120 min after adding reagents, and data were displayed after subtracting background values (Victor X4 Multi-Label Plate Reader).

Tallima H, & El Ridi R. (2023). Mechanisms of Arachidonic Acid In Vitro Tumoricidal Impact. Molecules, 28(4), 1727.

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Quantification of nSMase Activity

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Huh7.5.1 cells were infected with HCV or dengue virus at an MOI of 0.1. After 3 days, the culture medium was changed to exosome-depleted FBS medium. After 24 h, the cells were harvested, lysed in the RIPA Lysis Buffer System (Santa Cruz Biotechnology), and centrifuged at 12,000 × g for 20 min at 4°C. The protein content of the clear lysates was estimated using the Pierce BCA Protein Assay Kit, according to the manufacturer’s protocols (Thermo Fisher Scientific). Equal amounts of total protein were used for nSMase activity measurement using Sphingomyelinase Assay Kit according, to the manufacturer’s protocols (Abcam).

Kim O.K., Nam D.E, & Hahn Y.S. (2021). The Panx1/P2X4 pathway controls the secretion of miRNA-containing exosomes by HCV-infected hepatocytes. Hepatology (Baltimore, Md.), 74(6), 3409-3426.

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