Hep3B, HepG2, SK-Hep1, LM3, BEL-7404, SMMC-7721, and human hepatocyte LO2 cells were from Cell Bank of Type Culture Collection of Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Science, and cultured in Dulbecco's Modified Eagle's Medium (Gibco-Life Technologies) supplemented with 10% fetal bovine serum (FBS). Human umbilical vein endothelial cells (HUVECs) and human embryonic kidney cells (HEK-293T) were cultured in DMEM supplemented with 10% FBS. Hep3B, HepG2, SK-Hep1, LO2, and HEK-293T cells were authenticated by STR (short tandem repeats). BEL-7404, SMMC-7721 cells were identified by their morphological characteristics which were consistent with the report of establishment 13 . Cells were not contaminated by mycoplasma, and also not infected by bacteria or fungi. All cells were cultured in a humidified incubator with 5% CO2 at 37 °C. Plasmid DNA transfection assays were conducted when the confluence of incubated cells reached 60%-70%. For sulfatide treatment, cells were incubated at initial density 0.5x105 cells/mL and treated with 2 μM galactocerebroside (Gal-Cer) or sulfatide (Sigma, St. Louis, Missouri, USA).
Sulfatide
Manufactured by Merck Group
Sourced in United States
Sulfatide is a laboratory reagent used in biochemical research and analysis. It is a type of sulfated glycosphingolipid, which are important components of cell membranes and play roles in various biological processes. Sulfatide can be used for the detection and quantification of specific biomolecules in samples.
Automatically generated - may contain errors
Lab products found in correlation
Sphingomyelin, by Merck Group (2 mentions)
Tyloxapol, by Merck Group (1 mentions)
Hitrap q ff column, by GE Healthcare (1 mentions)
Cd3 clone ucht1, by BD (1 mentions)
Phosphatidylcholine pc, by Avanti Polar Lipids (1 mentions)
Sphingomyelin sm, by Avanti Polar Lipids (1 mentions)
Lysophosphatidylcholine lpc, by Avanti Polar Lipids (1 mentions)
Sulfatide, by Avanti Polar Lipids (1 mentions)
Lsrfortessa, by BD (1 mentions)
Streptavidin pe, by BD (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Pip strip, by Echelon Biosciences (1 mentions)
1 2 diacyl sn glycero 3 phospho l serine, by Merck Group (1 mentions)
Genetools software, by Syngene (1 mentions)
Membrane lipid strip, by Echelon Biosciences (1 mentions)
Mp4 25d2, by BioLegend (1 mentions)
Bvd2 23b6, by BioLegend (1 mentions)
Bvd2 21c11, by BioLegend (1 mentions)
α galactosylceramide α galcer, by Avanti Polar Lipids (1 mentions)
Ultrafast liquid chromatography system, by Shimadzu (1 mentions)
9 protocols using sulfatide
1
Sulfatide Treatment of Liver Cell Lines
2
Lipid-loaded CD1a Tetramer Preparation
3
Lipid Profiling of DRM Fractions
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Lipids from samples of DRM fractions were extracted as in the case of TLC. Then, lipids were spotted onto a Hybond-C membrane. The membrane was subsequently air-dried, blocked with 1% defatted milk in PBS for 1 h and overlaid with 2.5 μg/mL of recombinant toxin in blocking buffer overnight. After three washes with PBS supplemented with 0.1% Tween 20, bound toxin was detected using a polyclonal specific IgG raised in rabbit [17 (link)] and subsequent chemiluminescence, as in the case of western blots. To determine the presence of sulfatide in DRM gradients, a monoclonal antibody against sulfatide was used [51 (link)]. In some analyses, commercial membranes with spotted lipids were also used (Membrane lipid strips and PIP strips from Echelon Biosciences, USA) or, alternatively, commercial lipids were spotted onto Hybond C membranes. These lipids were: 1,2-Diacyl-sn-glycero-3-phospho-L-serine, sulfatide and a phosphoinositide mix (PIs) from bovine brain (all from Sigma). When indicated, signal intensity was determined using the GeneTools software (Syngene; Cambridge, UK).
4
Lipid Extraction and Separation Protocol
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Lipids from samples of the DRM fractions were extracted according to the Bligh and Dyer method [49 (link)]. During the extraction, acetic acid (0.057 M) was added to the water phase, in order to increase the recovery of acidic phospholipids [50 (link)]. Briefly, 960 μL of chloroform/methanol (1:2), 400 μL of chloroform and 200 μL of 0.1 M acetic acid were added, in that order, to 200 μL of sample. After shaking the mixture, the tubes were centrifuged (1,000 rpm, 5 min), and the organic phase was extracted and evaporated using Speed-Vac. When necessary, evaporated samples were resuspended in 25 μL of chloroform/methanol (4:1) and resolved on silica high-performance TLC plates using chloroform/methanol/water (68:28:4) when sphingolipid detection was required, or using chloroform/methanol/NH4OH/water (40:48:5:10) in the case of phosphoinositides (PIs). Standards (sulfatide, sphingomyelin or monophosphorylated PIs, all from Sigma-Aldrich) were resolved in parallel and staining of lipids was performed using primuline.
5
Lipid Stimulation of Monocyte-Derived Dendritic Cells
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Monocytes, Mo-DCs or CD1-transfected C1R cells were cultured with sulfatide (30–0.04 μg/mL, Sigma-Aldrich), GM1 (50–0.07 μg/mL, Sigma-Aldrich), α-Galactosylceramide (α-GalCer) (at 50 ng/mL or 50–3.12 ng/mL, Avanti polar lipids, Alabaster, AL, USA) or α-Gal-(1-2)-αGalCer (300–50 ng/mL). Lipids, with the exception of α-GalCer, were first dissolved in methanol or PBS 0.5% Tween 20 and then diluted in non-supplemented RPMI to have a maximum of 1% vehicle in culture. α-GalCer was resuspended in PBS and directly diluted in non-supplemented RPMI. After 4 h, an iNKT cell line or T cell clones were added and 40 h later, supernatants were collected for cytokine production determination by ELISA. The following antibody pairs from Biolegend were used: purified anti-human GM-CSF (BVD2-23B6) and biotinylated anti-human GM-CSF (BVD2-21C11); purified anti-human IL-4 (8D4-8) and biotinylated anti-human IL-4 (MP4-25D2).
6
In vitro Glucosylceramide Synthesis Assay
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Modified from Larsen et al.46 (link). Briefly, 60 µg of MDCK cell lysate was incubated with S202 in 100 mM Tris buffer (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA, 2 mM NAD, 100 μΜ UDP-glucose, 10 μΜ C6-NBD-Ceramide (Matreya), 35 μΜ DOPC, and 5 μΜ sulfatide (Sigma) at 37 °C for 1 h. The reaction was stopped with equal volume of acetonitrile and 50 μL was added to 100 µL of acetonitrile containing 100 ng/mL tolbutamide IS and centrifuged. Next, 70 µL of supernatant was mixed with 140 μL H2O before LC–MS analysis. NBD-Glucosylceramide quantification was performed on a Shimadzu ultra-fast liquid chromatography system (Shimadzu) coupled with an API 4000 triple quadrupole mass spectrometer in ESI positive mode (Applied Biosystems) and a Xbridge BEH130 CI 8, 100 mm × 4.6 mm i.d, 3.5 µm column (Waters). The mobile phase consisted of water and acetonitrile supplemented with 0.1% formic acid (v/v).
7
Investigating Integrin-Mediated Signaling Pathways
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Rabbit antibodies against phosphorylated FAK (pFAK, Tyr397, Tyr861 and Tyr925), phospho-paxillin-Y31, phospho-caveolin-1-Y14, phosphorylated integrin β3 (Tyr773/Tyr785) and non-phosphorylated FAK, paxillin and caveolin-1 were from Bioworld Technology (St. Louis Park, MN, USA). Phosphorylated FAK Tyr407 was from Abcam (Cambridge, UK). Phospho-Src-Y416 was from Cell Signaling Technology (Danvers, MA, USA). Antibodies against integrin αV, β3, Src and FAK inhibitor 14 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Integrin αVβ3 antibody LM609 was from Millipore (Temecula, CA, USA). Integrin β1 and β5 antibodies, PP2(1H-Pyrazolo[3,4-d]pyrimidin-4-amine, 3-(4-chlorophenyl)-1-(1,1-dimethylethyl)-) and SU6656((Z)-N, N-dimethyl-2-oxo-3-((4,5,6,7-tetrahydro-1H-indol-2-yl)methylene)indoline-5-sulfonamide) were from Merck (Darmstadt, Germany). Sulfatide, galactocerebroside, lactocerebroside and ganglioside (GM3) were from Sigma–Aldrich (St. Louis, MO, USA). Peptides GRGDSP and GRGESP with 99% purity were provided by China Peptides Co. (Shuzhou, China). Bodipy FL-C5 was from Molecular Probes (Carlsbad, CA, USA).
8
Lipid Standards for Chromatography
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AG-205 (cis-2-[[1-(4-Chlorophenyl)-1H-tetrazol-5-yl]thio]-1-(1,2,3,4,4a,9b-hexahydro-2,8-dimethyl-5H-pyrido[4,3-b]indol-5-yl)-ethanone) (cat# 6242) was obtained from Bio-Techne (Minneapolis, MN, USA), dissolved in DMSO (cat# 1.02931, Merck, Darmstadt, Germany) at a concentration of 10 mM, and stored in aliquots at −20 °C. Brefeldin A (BFA) (cat# B6542) was purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in ethanol at a final concentration of 5 mg/mL, and stored at −20 °C. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (cat# M5655) was purchased from Sigma-Aldrich, dissolved in water (10 mg/mL), and stored at −20 °C. Chloroform (cat# 7331) and methanol (cat# AE71) were obtained from Carl Roth (Karlsruhe, Germany). The following lipid standards were purchased from Sigma-Aldrich (St. Louis, MO, USA): cholesterol (cat# C8667), galactosylceramide (cat# C4905), phosphatidylcholine (cat# P3556), sphingomyelin (cat# S7004), and sulfatides (cat# S1006). C16 lactosylceramide (d18:1/16:0) (cat# 860576) and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (cat# 850702) were from Avanti Polar Lipids (Alabaster, AL, USA). All chromatographies were performed using silica gel 60 high-performance thin-layer chromatography (HPTLC) plates (cat# 1.05641, Merck, Darmstadt, Germany).
9
Activating Clotting Cascade In Vitro
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CTI was from Haematologic Technologies (Essex Junction, Vermont, United States); recombinant TF Innovin was from Dade Behring (Marburg, Germany); sulfatides and bovine serum albumin were from Sigma (St. Louis, Missouri, United States). Purified human FXII and FXI were obtained from Enzyme Research Laboratories (Stago, Leiden, the Netherlands). Recombinant tissue-type plasminogen activator (tPA) was from Boehringer Ingelheim (Alkmaar, the Netherlands). Synthetic phospholipids DOPS, DOPC, and DOPE (1,2-dioleoyl-
sn-glycero-3-phosphoserine, 1,2-dioleoyl-
sn-glycero-3-choline, and 1,2-dioleoyl-
sn-glycero-3-ethanolamine) were from Avanti Polar Lipids (Alabaster, Alabama, United States). Phospholipid vesicles (DOPS/DOPC/DOPE, 20/60/20 mol/mol/mol) were prepared by sonication, as described earlier.
24 (link)
Fluorogenic thrombin substrate, Z-Gly-Gly-Arg-aminomethyl coumarin (Z-GGR-AMC), was from Bachem (Bubendorf, Switzerland).
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CTI was from Haematologic Technologies (Essex Junction, Vermont, United States); recombinant TF Innovin was from Dade Behring (Marburg, Germany); sulfatides and bovine serum albumin were from Sigma (St. Louis, Missouri, United States). Purified human FXII and FXI were obtained from Enzyme Research Laboratories (Stago, Leiden, the Netherlands). Recombinant tissue-type plasminogen activator (tPA) was from Boehringer Ingelheim (Alkmaar, the Netherlands). Synthetic phospholipids DOPS, DOPC, and DOPE (1,2-dioleoyl-
sn-glycero-3-phosphoserine, 1,2-dioleoyl-
sn-glycero-3-choline, and 1,2-dioleoyl-
sn-glycero-3-ethanolamine) were from Avanti Polar Lipids (Alabaster, Alabama, United States). Phospholipid vesicles (DOPS/DOPC/DOPE, 20/60/20 mol/mol/mol) were prepared by sonication, as described earlier.
24 (link)
Fluorogenic thrombin substrate, Z-Gly-Gly-Arg-aminomethyl coumarin (Z-GGR-AMC), was from Bachem (Bubendorf, Switzerland).
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