Sphingosine 1 phosphate (s1p)

Manufactured by Cayman Chemical

Sourced in United States, Sweden

S1P is a laboratory reagent that functions as a sphingosine-1-phosphate standard. It is used for research and analytical purposes in various scientific fields.

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70 protocols using sphingosine 1 phosphate (s1p)

Thymocyte Migration and S1P Receptor Desensitization

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Enriched CD4 SP thymocytes were rested in migration medium (RPMI 1640/0.5% fatty acid–free BSA [Sigma-Aldrich]/10 mM Hepes) for 30 min at 37°C/5% CO₂. Migration through 5-µM Transwells (Corning) in response to 1 µg/ml CCL21 (R&D Systems), 0.3 µg/ml CXCL12 (PeproTech), or S1P (Cayman Chemical) was assessed after 3 h. For S1PR1 resensitization assays, thymocytes were first desensitized by incubation with a low (1–10 nM) or high (1,000 nM) concentration of S1P for 30 min. An aliquot of cells was incubated in medium alone (“no desensitization”). Cells were then either washed three times with warm RPMI 1640 and incubated in medium without S1P for 30 min (“resensitization”) or were not resensitized, i.e., washed once with RPMI 1640 just before the transwell assay (“desensitization”). To recover S1PR1 surface expression after desensitization with 1,000 nM S1P, thymocytes were incubated with CD45.1⁺ splenocytes as described previously (Kabashima et al., 2006 (link)). Chemotaxis to 50 nM S1P was then tested as above.

Willinger T., Ferguson S.M., Pereira J.P., De Camilli P, & Flavell R.A. (2014). Dynamin 2–dependent endocytosis is required for sustained S1PR1 signaling. The Journal of Experimental Medicine, 211(4), 685-700.

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Effects of S1P and S1PR3 Antagonist on SPK1

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The effects were compared of exogenous S1P and/or a S1PR3 antagonist (CAY10444) on SPK1 expression in TKE2 corneal epithelial cells or ocular fibroblasts. Both of these cell types express S1PR3 as detected by immunohistochemistry. They were grown to confluence in a 60 mm culture dish. Then the cells were incubated in a serum-free medium for another 16 h. Cells were cultured under the following conditions for another 24 h under one of the following four different conditions: (a) S1P (200 nM) (Cayman Chemical, Ann Arbor, MI, USA); (b) CAY10444 (a selective S1PR3 antagonist, 100 μM, (Cayman Chemical); (c) or both S1P and CAY10444 at the aforementioned concentrations. (d) serum- free culture served as the control. Ten dishes were prepared for each culture condition. RNA was extracted and processed for real-time qRT-PCR for VEGF-A. Mann–Whitney U test was used for data analysis.

Yasuda S., Sumioka T., Iwanishi H., Okada Y., Miyajima M., Ichikawa K., Reinach P.S, & Saika S. (2020). Loss of sphingosine 1-phosphate receptor 3 gene function impairs injury-induced stromal angiogenesis in mouse cornea. Laboratory Investigation; a Journal of Technical Methods and Pathology, 101(2), 245-257.

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Antibody and Inhibitor Assay Protocol

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Anti-Fpr2 (GM1D6) monoclonal antibody and anti-CRAMP (anticathelin-related antimicrobial peptide) (R-170) polyclonal antibody were from Santa Cruz (Santa Cruz, CA, USA). Anti-FGFR1, antiglutamine synthetase (anti-GS) and anti-Vimentin antibodies, and an FGFR antagonist PD 173074 were from Abcam (Cambridge, UK). The Fpr2 antagonist (WRW4) was purchased from Tocris Bioscience (R&D Systems, Minneapolis, MN, USA). Mouse CRAMP (NH2-ISRLAGLLRK GGEKIGEKLKKIGQKIKNFFQ KLVPQPE-OH) was synthesized by New England Peptide LLC (Gardner, MA, USA). Mouse b-FGF was purchased from Pepro Tech (Rocky Hill, NJ, USA). Sphingosine-1-phosphate (S1P) was purchased from Cayman Chemical Company (MI, USA). Antibodies specific for total ERK1/2, ERK1/2 phosphorylated at Tyr-204, phosphor (P)-p38 MAPK, and total p38 MAPK, were purchased from Cell Signaling Technology (Beverly, MA, USA). The IκB-α inhibitor BAY 11-7082 was purchased from Selleckchem (TX, USA). fMLF was obtained from Sigma-Aldrich.

Yu Y., Bao Z., Wang X., Gong W., Chen H., Guan H., Le Y., Su S., Chen K, & Wang J.M. (2017). The G-Protein-Coupled Chemoattractant Receptor Fpr2 Exacerbates High Glucose-Mediated Proinflammatory Responses of Müller Glial Cells. Frontiers in Immunology, 8, 1852.

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Synthesis and Characterization of 2-Hydroxyhexadecanoic Acid

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Sphingosine-1-phosphate (S1P) was purchased from Cayman Chemical (Ann Arbor, MI). Growth factor reduced Matrigel was obtained from BD Biosciences (Bedford, MA). Fertile chicken eggs were supplied by Charles River Labs (Wilmington, MA). All other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA) and used as received unless otherwise stated. 2-Hydroxyhexadecanoic acid was synthesized from 2-bromohexadecanoic acid per literature protocols [47 ].
NMR spectra were recorded on a Varian INOVA-400 spectrometer. Molecular weights and polydispersity of polymers were determined by gel permeation chromatography (GPC) on a Varian Prostar HPLC system equipped with two 5-mm PLGel MiniMIX-D columns and a PL-ELS2100 evaporative light scattering detector. Calibrations were performed with polystyrene standards (polymer laboratories). THF was used as the eluent at a flow rate of 0.3 mL/min.

Zhang J, & Song J. (2014). Amphiphilic degradable polymers for immobilization and sustained delivery of sphingosine 1-phosphate. Acta biomaterialia, 10(7), 3079-3090.

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Endothelial Cell Culture and Angiogenic Factors

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HepG2 (Riken Cell Bank, Ibaraki, Japan) was cultured in Dulbecco's modified Eagle's medium (DMEM; Nissui, Tokyo, Japan) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). Green fluorescent protein (GFP)-expressing human umbilical vein endothelial cells (GFP-HUVECs; Angio-Proteomie, Boston, MA, USA) were cultured in MCDB107 medium (Cell Science & Technology Institute, Miyagi, Japan) containing 10% FBS, 10 ng/mL human epidermal growth factor (Sigma-Aldrich), and 10 ng/mL human recombinant fibroblast growth factor-2 (ReproCELL, Kanagawa, Japan). The combination of angiogenic factors was based on a previous report [13] (link), including vascular endothelial growth factor (VEGF; Gibco, Frederick, MD, USA), monocyte chemotactic protein-1 (MCP-1; Gibco), sphingosine-1-phosphate (S1P; Cayman Chemical, Ann Arbor, MI, USA), and phorbol 12-myristate 13-acetate (PMA; Abcam, Cambridge, MA, USA). VEGF, MCP-1, and PMA were used at 75 ng/mL, and S1P at 500 nM.

Liu Y., Sakai S, & Taya M. (2016). Engineering tissues with a perfusable vessel-like network using endothelialized alginate hydrogel fiber and spheroid-enclosing microcapsules. Heliyon, 2(2), e00067.

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Differentiating PHH-derived Spheroids into Ductal Cells

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To assess the capacity of differentiation between PHH-derived spheroids and ductal cells, spheres were seeded in a serum-free medium, Williams Medium E, containing hHGF, bFGF and hIGF-1 (all 20 ng/mL) until they formed a monolayer. The medium was then changed to a transition and expansion medium (TEM) containing DMEM/F12 supplemented with insulin-transferrin-serine (ITS) (Cat# I3146, Sigma-Aldrich, Oakville, ON, Canada), with the following growth factors or small molecules: hEGF (20 ng/mL, Cat# 78006.1, Stemcell technologies, Vancouver, BC, Canada), hHGF (20 ng/mL, Cat# 100-39H, Peprotech, Cranbury, NJ, USA), Y27632 (10 μM, Cat# 10005583), CHIR99021 (3 μM, Cat# 13122-1), Sphingosine-1-phosphate (S1P) (1 μM, Cat#62570), lysophosphatidic acid (LPA) (5 μM, Cat#10010093-1) and A83-01 (1 μM, Cat# 9001799-5) (all from Cayman Chemical, Ann Arbor, MI, USA), for 7 days as described previously [16 (link)].

Yeganeh B., Yeganeh A., Malone K., Beug S.T, & Jankov R.P. (2023). Suspension-Induced Stem Cell Transition: A Non-Transgenic Method to Generate Adult Stem Cells from Mouse and Human Somatic Cells. Cells, 12(20), 2508.

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Inhibition of TGF-β1 Signaling Pathway

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Recombinant human TGF-β1 was purchased from Merck Millipore. TGF-β1 neutralizing antibody and control IgG antibody (Rabbit polyclonal IgG) were obtained from R&D systems. CAY10444, JTE-013, SB203580, sphingosine-1-phosphate (S1P), SKI-5C (CAY10621), SIS3, and W146 were obtained from Cayman Chemicals. A83-01 was purchased from Sigma-Aldrich. ALK5 inhibitor II was obtained from Enzo Life Sciences.

Beach J.A., Aspuria P.J., Cheon D.J., Lawrenson K., Agadjanian H., Walsh C.S., Karlan B.Y, & Orsulic S. (2015). Sphingosine kinase 1 is required for TGF-β mediated fibroblast-to-myofibroblast differentiation in ovarian cancer. Oncotarget, 7(4), 4167-4182.

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Functionalization of PLGA Substrates with S1P

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Fabricated PLGA substrates were functionalized with sphingosine 1-phosphate (S1P; Cayman Chemical, MI, USA) or fluorescein-S1P (Echelon Biosciences, UT, USA) using 3,4-dihydroxy-L-phenylalanine (DOPA; Sigma Aldrich). A 2 mg/mL working solution of was generated by dissolving DOPA in 10 mM tris(hydroxymethyl)aminomethane (Tris; Fisher Scientific, NH, USA) buffer with a pH of 8.5. DOPA working solution was combined with a 500 μM S1P stock solution for each sample to achieve the appropriate S1P concentration. Samples were then incubated at room temperature overnight on a rocker. Afterwards, the DOPA-S1P solution was aspirated and substrates were washed three times with PBS before being dried using nitrogen gas. Substrates not functionalized with S1P were incubated with DOPA only.

Tsui J.H., Janebodin K., Ieronimakis N., Yama D.M., Yang H.S., Chavanachat R., Hays A.L., Lee H., Reyes M, & Kim D.H. (2017). Harnessing Sphingosine-1-Phosphate Signaling and Nanotopographical Cues to Regulate Skeletal Muscle Maturation and Vascularization. ACS nano, 11(12), 11954-11968.

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Transwell Assay for Murine Hematopoietic Progenitor Cells

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Medium (650 μl of RPMI-1640 medium plus 0.5% BSA) containing SDF-1 (10 ng/ml; Pepro Tech, Rocky Hill, NJ, USA), sphingosine-1-phosphate (S1P; 0.1 μM; Cayman Chemical Company, Ann Arbor, MI, USA), ceramide-1-phosphate (C1P; 100 µM; Sigma-Aldrich), or ATP (0.25 ng/ml; Sigma-Aldrich) was added to the lower chambers of a Costar Transwell 24-well plate (Corning Costar, Cambridge, MA, USA). Aliquots of murine BM-MNCs resuspended in assay medium (1 × 10⁶ cells/100 μl) were loaded onto the upper chambers with 5-μm-pore filters and then incubated for 3 h (37 °C, 5% CO₂). An aliquot of cells from the lower chambers was harvested and counted by FACS analysis. The cells were gated according to their forward scatter (FSC) and side scatter (SSC) parameters and counted during a 30-s acquisition at a high flow rate. The remaining cells were resuspended in human methylcellulose base medium provided by the manufacturer (R&D Systems), supplemented with murine GM-CSF (25 ng/ml) and IL-3 (10 ng/ml), for determining the number of CFU-GM colonies. Cultures were incubated for 7 days (37 °C, 95% humidity, and 5% CO₂), at which time the colonies were counted under an inverted microscope [26 (link), 27 (link), 30 (link)].

Adamiak M., Bujko K., Cymer M., Plonka M., Glaser T., Kucia M., Ratajczak J., Ulrich H., Abdel-Latif A, & Ratajczak M.Z. (2018). Novel evidence that extracellular nucleotides and purinergic signaling induce innate immunity-mediated mobilization of hematopoietic stem/progenitor cells. Leukemia, 32(9), 1920-1931.

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Modulation of Sphingolipid Metabolism

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RSV, N-acetyl-L-cysteine (NAC), sulforaphane, curcumin, and quercetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Epigallocatechin gallate (EGCG) was purchased from Funakoshi Co. (Tokyo, Japan). Z-VAD-FMK, a pan-caspase inhibitor, was purchased from Promega (Madison, WI, USA). SPHK1 inhibitor SKI-II (described as SKI in the text), SPHK2 inhibitor ABC294640, GCS inhibitor PDMP, and sphingosine 1-phosphate (S1P) were purchased from Cayman Chemical (Ann Arbor, MI, USA). GT-11,¹³ a dihydroceramide desaturase (DES1) inhibitor, was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA).

Inoue C., Sobue S., Mizutani N., Kawamoto Y., Nishizawa Y., Ichihara M., Takeuchi T., Hayakawa F., Suzuki M., Ito T., Nozawa Y, & Murate T. (2020). Vaticanol C, a phytoalexin, induces apoptosis of leukemia and cancer cells by modulating expression of multiple sphingolipid metabolic enzymes. Nagoya Journal of Medical Science, 82(2), 261-280.

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