All chemicals are molecular biology-grade (SIGMA-Aldrich) unless specified, common solvents were from Merck. High performance silica gel-precoated thin-layer plates (HPTLC Kieselgel 60) were purchased from Merck GmbH. [3-3H]Sphingosine (specific radioactivity 19.8 Ci/mmol) was provided by PerkinElmer Life Sciences.
3 3h sphingosine
Manufactured by PerkinElmer
Sourced in United States
[3-3H]sphingosine is a radioactively labeled compound used in research applications. It is a tritium-labeled form of the lipid sphingosine, which is a key component of cell membranes and involved in various cellular signaling processes.
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Lab products found in correlation
4 protocols using 3 3h sphingosine
1
Radioactive Sphingosine Synthesis
2
Sphingolipid Analysis via Metabolic Labeling
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Sphingolipid analysis was carried out through cell metabolic labelling with [3-3H]sphingosine (PerkinElmer, Waltham, MA, USA), as previously reported.20 (link) Ganglioside and neutral sphingolipid extracts were analysed by HPTLC carried out with the solvent systems chloroform/methanol/0.2% CaCl2 55 : 45 : 6 (v/v) and chloroform/methanol/water 110 : 40 : 6 (v/v), respectively.
3
Radiolabeling of Cell Sphingolipids
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The metabolic radiolabeling of cell sphingolipids was performed as previously described by Riboni et al. [18 (link)]. Briefly, [3-3H]-sphingosine (D-erythro > 97%, 50 μCi, 1.85 MBq, PerkinElmer) was dissolved in DMEM-low glucose with 10% FBS to a final concentration of 2.4 nM sphingosine, corresponding to 110.000 dpm/ml radioactivity. The medium was added to the cells and incubated for 2 hours (pulse) at 37°C, then it was replaced with DMEM-low glucose with 10% FBS without [3H]-sphingosine for 48 hours (chase). After the incubation, cells were harvested by cell scraping in phosphate-buffered saline (PBS). Cell suspensions were frozen and lyophilized.
4
Sphingolipid Analysis via Metabolic Labeling
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Sphingolipid analysis was carried out through cell metabolic labeling with [3-3H]sphingosine (PerkinElmer, Waltham, MA, USA) [28 (link)]. In order to assay the hypothesis that Neu5Gc-glicans could be incorporated from the culture medium and then employed for the synthesis of GM3, before [3-3H]sphingosine labeling, melanoma L6 cells were pre-incubated in the reduced-serum medium OptiMEM (Life Technology, Carlsbad, CA, USA) for 5 days.
Ganglioside and neutral sphingolipid extracts were analyzed by HPTLC carried out with the solvent systems chloroform/methanol/0.2% CaCl2 55:45:6 (v/v) and chloroform/methanol/water 110:40:6 (v/v), respectively. To separate Neu5Gc-GM3 from Neu5Ac-GM3, HPTLC was carried out using the solvent system chloroform/methanol/0.2% CaCl2/5 N NH3 50:42:6:4 (v/v). The sphingolipid pattern was determined and quantified by radiochromatoscanning (Betaimager 2000, Biospace, Paris, France) [28 (link), 29 (link)].
Endogenous sphingolipid analysis performed to standardize metabolic labeling was performed as previously described [30 (link)].
Ganglioside standards were kindly given by Prof. Sonnino, University of Milan.
Ganglioside and neutral sphingolipid extracts were analyzed by HPTLC carried out with the solvent systems chloroform/methanol/0.2% CaCl2 55:45:6 (v/v) and chloroform/methanol/water 110:40:6 (v/v), respectively. To separate Neu5Gc-GM3 from Neu5Ac-GM3, HPTLC was carried out using the solvent system chloroform/methanol/0.2% CaCl2/5 N NH3 50:42:6:4 (v/v). The sphingolipid pattern was determined and quantified by radiochromatoscanning (Betaimager 2000, Biospace, Paris, France) [28 (link), 29 (link)].
Endogenous sphingolipid analysis performed to standardize metabolic labeling was performed as previously described [30 (link)].
Ganglioside standards were kindly given by Prof. Sonnino, University of Milan.
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