3h sphingosine

Manufactured by PerkinElmer

Sourced in Australia

[3H]-sphingosine is a radioactive tracer compound that can be used to study the metabolism and distribution of sphingosine, a bioactive lipid, in biological systems. It is a tool for researchers to investigate the role of sphingosine and sphingolipid pathways in various cellular processes and disease states.

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Lab products found in correlation

High performance tlc silica gel 60 plates, by Merck Group (1 mentions)

Close spelling variants of this product

[3-3H]sphingosine (4 citations) [3-3H] D-erythro-sphingosine (2 citations)

4 protocols using 3h sphingosine

Extracellular S1P Formation Assay

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Rate of extracellular S1P formation from intact vector control ‘low’, SK2 ‘low’ and SK2 ‘high’ NIH3T3 stable cell lines was determined essentially as previously described [47 (link)]. Briefly, cells were seeded in equal numbers at 80% confluence (in 20 cm² dishes), and media was replaced with DMEM containing 0.5% DBS and incubated for a further 16 h. Cells were then labelled with 0.5 μCi of [³H]-sphingosine (Perkin-Elmer, Rowville, VIC, Australia) for 30 min, after which the conditioned media was collected. Extracellular [³H]-S1P generated in the conditioned medium was extracted and analyzed by scintillation counting.

Neubauer H.A., Pham D.H., Zebol J.R., Moretti P.A., Peterson A.L., Leclercq T.M., Chan H., Powell J.A., Pitman M.R., Samuel M.S., Bonder C.S., Creek D.J., Gliddon B.L, & Pitson S.M. (2016). An oncogenic role for sphingosine kinase 2. Oncotarget, 7(40), 64886-64899.

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Radioactive Pulse-Chase Assay for Lipid Analysis

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Radioactive pulse‐chase assay was performed as described earlier (D'Angelo et al, 2013 (link)). Briefly, HeLa‐M cells or human fibroblasts wi‐26 or GRASP55 KO cells were pulse labeled for 2 h with [³H]‐sphingosine (final concentration of 0.1 μCi/ml; Perkin Elmer) in serum‐free DMEM containing 1% fatty acid‐free BSA followed by a chase for indicated times in complete media. Lipids were then extracted from the cells, resuspended in chloroform, spotted onto silica‐gel high‐performance TLC (HPTLC) plates (Merck, Germany), resolved using a mixture of chloroform, methanol, and water (65:25:4 v/v/v) and quantified using GINA® (Raytest, Germany) software analysis.

Pothukuchi P., Agliarulo I., Pirozzi M., Rizzo R., Russo D., Turacchio G., Nüchel J., Yang J., Gehin C., Capolupo L., Hernandez‐Corbacho M.J., Biswas A., Vanacore G., Dathan N., Nitta T., Henklein P., Thattai M., Inokuchi J., Hsu V.W., Plomann M., Obeid L.M., Hannun Y.A., Luini A., D’Angelo G, & Parashuraman S. (2021). GRASP55 regulates intra‐Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis. The EMBO Journal, 40(20), e107766.

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Quantification of Extracellular S1P Release

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The rate of extracellular S1P formation from intact cells was determined as previously described [52 (link)]. Briefly, cells were seeded into 6-well plates and were grown in MEM containing 10% FBS overnight. Cells, at 80% confluence, were then labeled with 0.5 µCi of [³H]-sphingosine (Perkin-Elmer, Rowville, VIC, Australia), delivered in serum-free media (7.5% final serum concentration on cells), for 30 min. The conditioned media were then collected and extracellular [³H]-S1P generated in the conditioned medium was extracted via a modified Bligh–Dyer extraction [52 (link)], and analyzed by scintillation counting. Analyses were performed in quadruplicate with data normalized to cell number.

Neubauer H.A., Tea M.N., Zebol J.R., Gliddon B.L., Stefanidis C., Moretti P.A., Pitman M.R., Costabile M., Kular J., Stringer B.W., Day B.W., Samuel M.S., Bonder C.S., Powell J.A, & Pitson S.M. (2018). Cytoplasmic dynein regulates the subcellular localization of sphingosine kinase 2 to elicit tumor-suppressive functions in glioblastoma. Oncogene, 38(8), 1151-1165.

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Sphingosine Labeling Assay Protocol

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Sphingosine labeling assay was performed as previously described (Nakahara et al., 2012 (link)). Cells were incubated for 12 h at 37°C with DMEM containing charcoal-treated 10% FCS and for another 12 h with DMEM. Cells were then labeled with 0.6 μCi of [³H]sphingosine (PerkinElmer Life Sciences, Waltham, MA) for 4 h at 37°C. Lipids were extracted, separated by normal-phase TLC on Silica Gel 60 high-performance TLC plates (Merck Millipore, Billerica, MA) with 1-butanol/acetic acid/water (3:1:1 [vol/vol]), and visualized by fluorography.

Yamamoto S., Yako Y., Fujioka Y., Kajita M., Kameyama T., Kon S., Ishikawa S., Ohba Y., Ohno Y., Kihara A, & Fujita Y. (2016). A role of the sphingosine-1-phosphate (S1P)–S1P receptor 2 pathway in epithelial defense against cancer (EDAC). Molecular Biology of the Cell, 27(3), 491-499.

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