n-Dodecyl-β-D-maltopyranoside (DDM) and n-decyl-β-D-maltopyranoside (DM) anagrade were obtained from Anatrace Inc or Glycon (Germany). Isopropyl-β-D-thiogalactoside (IPTG) was obtained from Formedium and (tris(2-carboxyethyl)phosphine (TCEP) from Thermo Scientific Ltd. The S-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate (MTSSL) spin label was obtained from Toronto Research chemicals, Toronto. Phospholipids, E. coli Polar Extract, PC, PE, PG, LPC 18:1 and LPC (14:0) were purchased from Avanti Polar Lipids. All other chemicals unless otherwise stated were obtained from Sigma. Mutants were generated with the Stratagene QuickChange™ protocol as described previously12 ,19 (link).
Lpc 18 1
Manufactured by Avanti Polar Lipids
Sourced in United States
LPC 18:1 is a lipid product offered by Avanti Polar Lipids. It is a lyso-phosphatidylcholine with a stearoyl (18:1) fatty acid chain. The core function of this product is to serve as a laboratory reagent for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
N dodecyl β d maltopyranoside ddm, by Anatrace (2 mentions)
Isopropyl β d thiogalactoside, by Formedium (2 mentions)
S 2 2 5 5 tetramethyl 2 5 dihydro 1h pyrrol 3 yl methyl methanesulfonothioate mtssl spin label, by LGC (2 mentions)
N decyl β d maltopyranoside anagrade, by Anatrace (2 mentions)
E coli polar extract, by Avanti Polar Lipids (2 mentions)
Lpc 14 0, by Avanti Polar Lipids (2 mentions)
Lpc 18 0, by Avanti Polar Lipids (2 mentions)
Lysophosphatidylcholine, by Avanti Polar Lipids (1 mentions)
Lpc 16 0, by Avanti Polar Lipids (1 mentions)
Lpc 18 2, by Avanti Polar Lipids (1 mentions)
Lpc 20 0, by Avanti Polar Lipids (1 mentions)
Lsrfortessa x 50 flow cytometer, by BD (1 mentions)
Facs staining buffer, by BioLegend (1 mentions)
Anti cd3 antibody okt3, by BioLegend (1 mentions)
Choline oxidase, by MP Biomedicals (1 mentions)
Amplex red, by Thermo Fisher Scientific (1 mentions)
Flexstation 3 multi mode microplate reader, by Molecular Devices (1 mentions)
Cacl2, by Carl Roth (1 mentions)
Sodium chloride (nacl), by Carl Roth (1 mentions)
α d glucose, by Merck Group (1 mentions)
7 protocols using lpc 18 1
1
Membrane Protein Preparation and Labeling
2
Obtaining Lysophosphatidylcholine Lipids
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Lysophosphatidylcholines (LPC
16:0, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:0, and LPC 22:0) were obtained
from Avanti Polar Lipids (Alabaster, AL). All solvents and organic
reagents were of the highest grade commercially available.
16:0, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:0, and LPC 22:0) were obtained
from Avanti Polar Lipids (Alabaster, AL). All solvents and organic
reagents were of the highest grade commercially available.
3
CD1a Lipid Tetramer Staining Protocol
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Biotinylated human CD1a monomers (NIH Tetramer Core Facility) were produced in HEK293-derived cell lines (36 (link), 67 (link)). CD1a (10 ug) was treated with a 100X molar excess of LPC 18:1 or LPC 18:0 (Avanti Polar Lipids) in Tris Buffer saline containing 0.25% CHAPS or vehicle alone (mock) for 16 h at 37 °C, and tetramerised with PE Streptavidin (High Concentration; BioLegend) at a molar ratio of 5:1. T cells (<1x106) were washed twice in FACS staining buffer (BioLegend) at room temperature and stained with 0.5 µl tetramer in 20 µl FACS staining buffer at 37 °C for 30 min with gentle shaking. Anti-CD3 antibody (OKT3; 0.1 µg in 10 µl; BioLegend) was added to the cells and incubated for an additional 10 min at 37 °C with gentle shaking. Tetramers and anti-CD3 antibody were removed before staining surface markers CD3 (UCHT1; BioLegend), CD4, CD8 and Zombie Fixable Viability dyes (BioLegend) for 15 min at 4°C. Cells were washed once and resuspended in FACS buffer and ready for requisition using LSRFortessa X-50 flow cytometer (BD Biosciences) and further analyzed with FlowJo (FlowJo LLC) software.
4
Characterizing ATX-Inhibitor Interactions
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First, we used the same FS-3 assay as described above, but instead of plasma 10 μL of human recombinant ATX (3, 10 and 30 nM) (Daresbury Proteins Ltd, Warrington, UK) was incubated with FS-3 substrate ± IND (30–300 μM).
In addition, the effect of IND on human recombinant ATX-induced hydrolysis of LPC 18:1 was assessed with the Amplex Red choline release assay. ATX was generated in-house, as described before [43 (link)]. Triplicate wells were loaded with 60 μL of reaction cocktail in ATX assay buffer (50 mM TRIS, 150 mM NaCl, 5 mM CaCl2, 30 μM BSA, pH 7.4), with of 10 μM Amplex Red, 1 U/mL horeseradish peroxidase (both from Thermo Fisher Scientific, Waltham, MA, USA) and 0.1 U/mL choline oxidase (MP Biomedicals, Irvine, CA, USA), resulting in an overall concentration of 100 μM LPC 18:1 (Avanti Polar Lipids, Alabaster, AL, USA) with 10 nM ATX ± IND (30–300 μM). The fluorescence (λexcitation = 560 nm and λemission = 590 nm) was recorded every 2 min by a FlexStation 3 Multi-Mode Microplate Reader for 240 min (Molecular Devices, San Jose, CA, USA).
In addition, the effect of IND on human recombinant ATX-induced hydrolysis of LPC 18:1 was assessed with the Amplex Red choline release assay. ATX was generated in-house, as described before [43 (link)]. Triplicate wells were loaded with 60 μL of reaction cocktail in ATX assay buffer (50 mM TRIS, 150 mM NaCl, 5 mM CaCl2, 30 μM BSA, pH 7.4), with of 10 μM Amplex Red, 1 U/mL horeseradish peroxidase (both from Thermo Fisher Scientific, Waltham, MA, USA) and 0.1 U/mL choline oxidase (MP Biomedicals, Irvine, CA, USA), resulting in an overall concentration of 100 μM LPC 18:1 (Avanti Polar Lipids, Alabaster, AL, USA) with 10 nM ATX ± IND (30–300 μM). The fluorescence (λexcitation = 560 nm and λemission = 590 nm) was recorded every 2 min by a FlexStation 3 Multi-Mode Microplate Reader for 240 min (Molecular Devices, San Jose, CA, USA).
5
Membrane Protein Preparation and Labeling
6
Lipid Preparation for Cellular Studies
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LPC 18:1 (Avanti Polar Lipids) in chloroform was aliquoted under argon, evaporated under nitrogen until dry and stored at −20°C under argon until use. LPC aliquots were dissolved in PBS to yield a stock solution (3 mM) and used fresh for every experiment. NaCl, KCl and CaCl2 were from Roth, KH2PO4 and NaHCO3 from Merck (Darmstadt, Germany), MgSO4 was from Fluka and α-D-Glucose was from Sigma-Aldrich.
7
LPS-Induced Inflammation: Lipid Mediators Analysis
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LPS (from E. coli O26 by phenol extraction) was purchased from FUJIFILM Wako (Tokyo, Japan) . LPC18:1 and LPE18:1 were purchased from Avanti Polar Lipids (Alabaster, AL, USA) . CD11b antibody (ab133357) and apocynin were purchased from Abcam (Cambridge, MA, USA) . Iba1 antibody (MABN92) was purchased from Sigma-Aldrich (St. Louis, MO, USA) . β-actin (sc-47778) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) .
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