Sphingosine‐1‐phosphate (S1P) was purchased from Enzo Life Science, (USA Cat# BML‐SL140‐000). Sphingosine kinase inhibitor (SK1‐II) was purchased from Tocris (Cat# 2097). LY2109761 was purchased from Sigma Aldrich (Italy, Cat# SML2051). Periodic acid‐Schiff was purchased from Sigma Aldrich (St Louis, USA Cat # 395B‐1KT). TGF‐β was purchase by R&D (USA, Cat #DB100B). Anti‐actin alpha SMA from Sigma Aldrich (Cat # A5228). Anti‐IL33 was purchased from ThermoFisher (Italy Car# AF3626). Anti‐FGF2 was purchased from Santa Cruz Technology (USA Cat# sc‐74412). Diamino‐benzidinic acid system was purchased from Sigma Aldrich (Italy Cat# D3939). OCT medium was purchased from Tissue‐Tek OCT (Pella Inc. Italy, Cat # 27050). High‐capacity cDNA kit and SYBR Green Real‐Time PCR Master Mix were purchased from ThermoFisher Scientific (Monza, Italy Cat # 4368814 and 4385612). CD90 PE mouse/rat and CD326 APC mouse were purchase from a Milteny iBiotec (Bologna, Italy Cat# 130‐094‐528 and #130‐096‐417). TGF‐β1 protein (Cat #ab50036), anti‐wide spectrum Cytokeratin (Cat #ab9377), rabbit anti‐mouse, Vimentin (Cat # ab92547), goat anti‐mouse IgG H&L FITC GtxMu‐003‐D (Cat # ab96885594) and goat anti‐rabbit IgG H&L DyLight (Cat #ab96886) were purchased from AbCAM (Cambridge, UK). Other compounds (e.g., ovalbumin, DAPI, collagenase) were purchased from Sigma Aldrich (Italy).
Sphingosine 1 phosphate (s1p)
Manufactured by Enzo Life Sciences
Sourced in United States, United Kingdom
S1P is a laboratory product offered by Enzo Life Sciences. It is a chemical compound that functions as a signaling molecule in biological systems. The core function of S1P is to serve as a tool for researchers studying cellular processes and signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Jte 013, by Bio-Techne (3 mentions)
Lysophosphatidic acid, by Merck Group (2 mentions)
Vpc23019, by Avanti Polar Lipids (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Luteolin, by Merck Group (2 mentions)
Jte 013, by Cayman Chemical (2 mentions)
Dihydro s1p, by Enzo Life Sciences (2 mentions)
Formyl methionyl leucyl phenylalanine (fmlp), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Sybr green real time pcr master mix, by Thermo Fisher Scientific (1 mentions)
Collagenase, by Merck Group (1 mentions)
Periodic acid schiff, by Merck Group (1 mentions)
Vimentin, by Abcam (1 mentions)
Ly2109761, by Merck Group (1 mentions)
Anti fgf 2, by Santa Cruz Biotechnology (1 mentions)
Tgf β, by R&D Systems (1 mentions)
High capacity cdna kit, by Thermo Fisher Scientific (1 mentions)
Facsaria, by BD (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
28 protocols using sphingosine 1 phosphate (s1p)
1
Molecular Regulation of Tissue Fibrosis
2
Traumatic Brain Injury and Immune Response
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Blood samples were collected from tail vein in 1 and 4 h after TBI to assess plasma cortisol and circulating lymphocytes or 4 h after hydrocortisone injection (Sigma, 10 mg/kg) to confirm suppressive effects of cortisol on peripheral leukocytes. In separate groups of mice, TBI was induced as above, immediately followed with i.p. injection of either sphingosine 1-phosphate (S1P) (Enzo Life Sciences, 5 μM/kg) or rolipram (Sigma, 30 μM/kg), and blood samples were collected 1 h later. Cells were pelleted, suspended, and treated with ammonium-chloride-potassium (ACK) buffer to lyse erythrocytes. The cells were then counted and stained with PE-anti-CD3 antibody for T cells, APC-anti-CD19 antibody for B cells, FITC-anti-Ly6G antibody for neutrophils, or PE-Cy7-anti-F4/80 antibody for monocytes, followed by flow cytometry analysis on BD FACSAria.
3
Purification and Use of SHH, PUR, S1P
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Recombinant SHH (SHH) was purified as previously described [28 (link)]. Purmorphamine (PUR) was purchased from EMD Millipore (#540220) and dissolved in DMSO at 10 mM. Sphingosine-1-phosphate (S1P) was purchased from Enzo Life Sciences (BML-SL140-001) and dissolved in fatty acid-free bovine serum albumin (BSA) at 4 mg/ml. Forskolin (FSK, Cat. #F6886), 3-isobutyl-1-methylxanthine (IBMX, Cat. #I5879), and Pertussis Toxin from Bordetella pertussis (PTX, Cat. #P280) were purchased from Sigma-Aldrich.
4
Fibroblast Activation and Regulation
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Normal human foreskin fibroblasts (NHDF, Promocell C-123000, lot 0062308), normal human lung fibroblasts (NHLF #2512, Lonza, lot 543644) and normal human cardiac atrial fibroblasts (NHCF, Lonza #2903, lot 214476), were cultivated on gelatin coated plates in complete cell growth medium FBM supplemented with FGM-2 or FGM-3 SingleQuots (Lonza) up to passage 4. Starvation medium was prepared from FBM (Lonza) and contained 0.1% fatty acid free bovine serum albumin (BSA,Calbiochem) and penicillin/streptomycin. Cells were maintained at 5% CO2 and 37°C. Cells were treated with the following stimuli/compounds: TGFβ1 (Peprotech), Rho inhibitor I and Rho activator II (Cytoskeleton), lysophosphatidic acid (LPA), latrunculin B, thrombin, 2-deoxy-D-glucose (Sigma Aldrich) and sphingosine-1-phosphate (S1P, Enzo Life Sciences).
5
Angiogenic Signaling Pathway Assay
6
Sphingolipid Signaling Molecules Analysis
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Sphingosine 1-phosphate, and D-erythro (S1P) were purchased from Enzo Life Sciences (Plymouth Meeting, PA). C17-Sphingosine 1-phosphate (C17-S1P), lysophosphatidic acid (LPA), lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI), and VPC23019 were purchased from Avanti Polar Lipids Inc (Alabaster, AL). Pertussis toxin (PTX) was obtained from Wako Pure Chemical Industries (Osaka, Japan).
S1P, LPA, LPS and LPI were dissolved in methanol. Just before use, the methanol was evaporated and the reagents were resolved in PBS containing 0.4% fatty acid-free BSA (Sigma-Aldrich Co., St. Louis, MO). C17-S1P was dissolved in methanol. VPC23019 and PTX were dissolved in DMSO.
S1P, LPA, LPS and LPI were dissolved in methanol. Just before use, the methanol was evaporated and the reagents were resolved in PBS containing 0.4% fatty acid-free BSA (Sigma-Aldrich Co., St. Louis, MO). C17-S1P was dissolved in methanol. VPC23019 and PTX were dissolved in DMSO.
7
Ceramide-Induced Cell Death Mechanisms in SH-SY5Y Cells
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SH-SY5Y cells were treated with cell-permeable, biologically active ceramide, C2-ceramide, at a 10 to 50 μM concentration for various times up to 24 h. In most of the experiments, the cells were treated with the following compounds: PJ-34 (20 μM), PARP-1 inhibitor; α-pifithrin (20 μM), p53 inhibitor; cyclosporine A (2 μM), inhibitor of the permeability transition pore; UO126 (1 μM), ERK1/2 kinase inhibitor; SP600125 (5 μM), JNK kinase inhibitor; sphingosine-1-phosphate (1 μM), LY294002 (50 μM), PI3-K inhibitor and Z-DEVD-FMK (100 μM), caspase-3 inhibitor added 1 h before incubation with C2-ceramide (25 μM). Our data showed that most of these compounds had no effect on cell viability in the control condition, with the exception of PARP-1 inhibitor, JNK kinase inhibitor and PI3-K inhibitor, which significantly decreased SH-SY5Y cell survival (by about 20 %, PJ-34, SP600125; 35 %, LY294002). The above-mentioned compounds were purchased from the following: C2-ceramide and sphingosine-1-phosphate from Enzo Life Sciences, LY294002 from Cell Signalling Technology, PJ-34, α-pifithrin, SP600125, U0126 and cyclosporine A from Sigma-Aldrich, Z-DEVD-FMK from Tocris Bioscence.
8
Preparation and Administration of S1P
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S1P (Enzo Life Sciences) was prepared according to the manufacturer’s instructions. Briefly, S1P was dissolved in methanol (0.5 mg/ml) and aliquoted, followed by evaporation of the solvent under a stream of nitrogen to deposit a thin film on the inside of the tube. Prior to use, the aliquots were resuspended in PBS with 4 mg/ml bovine serum albumin (BSA) to a final concentration of S1P at 500 μM. The S1P or the vehicle was i.p. injected into the mice at a dosage of 200 μl per mouse immediately after TBI.
9
IgE Measurement and Modulation
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Dinitrophenyl- (DNP-) specific mouse IgE was a generous gift from Dr. Daniel Conrad (VCU, Richmond, VA). DNP-human serum albumin (HSA) and ionomycin were obtained from Sigma-Aldrich (St Louis, MO). S1P was purchase from Enzo Life Sciences (Farmingdale, NY). JTE-013 and CYM-5442 were purchased from Tocris/Bio-Techne (Minneapolis, MN).
10
Stimulation of Lipid Signaling Pathways
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Cells were grown on standard cell culture plates in RPMI 1640 medium supplemented with 10% FBS until ∼80% confluent. Cells were serum‐starved for 24 h in serum‐free RPMI 1640 medium, then incubated with 10 μM 18:1‐LPA (Echelon Biosciences, UT), 10 μM S1P (Enzo Life Sciences, NY), and/or 1 μM TUG‐891 (Millipore Sigma, MO) for the indicated times. LPA and S1P were delivered as 1000X stock solutions prepared in 4 mg/mL fatty acid‐free BSA. Sources for other species of LPA were: 18:0‐LPA (Avanti Polar Lipids), 18:2‐LPA, and 18:3‐LPA (Echelon Biosciences, UT). Cells were rinsed twice with ice‐cold phosphate‐buffered saline (PBS), harvested by scraping into 1 mL ice‐cold PBS, collected by microcentrifugation for 10 min at 4°C, and resuspended in ice‐cold lysis buffer (20 mM HEPES [pH = 7.4], 1% Triton X‐100, 50 mM NaCl, 2 mM EGTA, 5 mM β‐glycerophosphate, 30 mM sodium pyrophosphate, 100 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin). Insoluble debris was removed following microcentrifugation. Protein concentrations of the supernatant “whole‐cell extracts” were determined using a bicinchoninic acid assay (Pierce).
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