*4.6. Cell Sphingolipid Labelling with [1-3H]-Sphingosine*

[1-3H]-sphingosine was administered as tracer in non-bioactive concentration for 2 h (pulse) followed to 96 h (chase), to allow steady state metabolic labelling of all cell SLs [39]. Briefly, [1-3H]-sphingosine dissolved in methanol was transferred into a sterile glass tube, dried under a nitrogen stream and then solubilized in an appropriate volume of pre-warmed (37 ◦C) cell culture medium to obtain a final concentration of 30 nM. The correct solubilization was verified by measuring the radioactivity associated with an aliquot of the medium using a β-counter (PerkinElmer, Waltham, MA, USA). After 2 h of incubation (pulse) the medium was removed, and the cells were incubated for 96 h (chase) in fresh culture medium without radioactive sphingosine. After chase, cells were collected, lyophilized and subjected to lipid extraction and SLs analysis. Total lipids from lyophilized cells were extracted with chloroform:methanol:water 20:10:1 by volume, followed by a second extraction with chloroform:methanol: 2:1 by volume. The radioactivity associated with total lipid extract, was evaluated by liquid scintillation, using a β-counter system (PerkinElmer).

[ 3H]SLs of total extracts were separated by high performance thin layer chromatography (HPTLC), using the solvent system chloroform:methanol:water 110:40:6 by volume. [3H]SLs were identified by digital autoradiography using TRacer system (Biospace Lab) and quantified with M3vision software. The lipid identification was performed using purified radioactive standards.

#### *4.7. Lipid Analysis by SFC-MS/MS*

Quantitative analysis of glucosylceramide, galactosylceramide, glucosylsphingosine and galactosylsphingosine was performed by Lipidomics Shared Resources Analytical Unit (Medical University of South Carolina, Charleston, SC, USA) [40]. Briefly, quantitative analysis of sphingolipids is based on eight-point calibration curves generated for each target analyte. The synthetic standards, along with a set of internal standards, were spiked into an artificial matrix and subjected to an identical extraction procedure as the biological samples. These extracted standards were then analyzed by the SFC-MS/MS system operating in positive MRM mode employing a gradient elution. Peaks for the target analytes and internal standards were recorded and processed using the instrument's software system. The calibration curve for a particular analyte was generated by plotting the analyte/internal standard peak area ratio against analyte concentrations. Any sphingolipid for which no standards were available was quantitated using the calibration curve of its closest counterpart. Separation of galactosylceramide and glucosylceramide was performed by SFC-MS/MS. The equipment consisted of a Waters UPC2 system coupled to a Thermo Scientific Quantum Access Max triple quadrupole mass spectrometer, equipped with an ESI (electrospray ionization) probe operating in the multiple reaction monitoring positive ion mode tuned and optimized for the Waters UPC2 system. Chromatographic separations were obtained utilizing carbon dioxide gas and 1 mM ammonium formate in 0.2% formic acid in the methanol mobile phase.

Analytical results were expressed as pmoles of lipid/mg of total cellular proteins. Data were the mean of two independent triplicate experiments.

#### **Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/10/ 3099/s1.

**Author Contributions:** A.M. performed biochemical and real-time PCR experiments, analyzed data and contributed to the writing, reviewing and editing of the manuscript; M.S. performed biochemical experiments and reviewed the manuscript; A.D. had a critical input in the interpretation of results, writing, reviewing and editing the manuscript; C.V. performed the Sanger sequencing confirmation of the LCLs and real-time PCR experiments, and reviewed the manuscript; S.S. had an input in the supervision and interpretation of the analyses related to the evaluation of the SL pattern; E.C. contributed to the preparation of the figures and writing of the discussion; M.P. and E.Z.-P. contributed to the patient samples as well as the clinical evaluation and reviewed the manuscript; M.A. and N.L. had a critical input in the conceptualization of the study, writing, reviewing and editing the manuscript; K.C. contributed in conceiving the study and reviewing the manuscript.

**Funding:** This study was funded by the Cyprus Institute of Neurology & Genetics and from the University of Milano.

**Acknowledgments:** We would like to thank the patients and healthy individuals who participated in this study.

**Conflicts of Interest:** The authors declare no conflict of interest.
