Phosphatidylcholine pc

Manufactured by Avanti Polar Lipids

Sourced in United States

Phosphatidylcholine (PC) is a naturally occurring lipid that serves as a key component of cell membranes. It is a phospholipid that can be isolated and purified for use in various laboratory applications.

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20 protocols using phosphatidylcholine pc

Lipid Extraction from Small Extracellular Vesicles

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The modified Bligh and Dyer method was used for lipid extraction as described previously.¹⁹, ²⁰ Phosphatidylcholine (PC) (12:0_12:0) (Avanti Polar Lipids) was used as an internal standard. In a glass tube, 40 μL of each sEV sample was mixed with 0.34 mL of methanol, 0.17 mL of chloroform, 0.14 mL of 0.322 M glacial acetate, and 0.182 nmol of PC(12:0_12:0). The solution was incubated for 10 min at 24°C, mixed with 0.17 mL of chloroform and 0.17 mL of 0.322 M glacial acetate, and allowed to separate into two phases. The lipid layer (lower phase) was separated from the aqueous layer by centrifugation at 720× g for 10 min at 24°C and transferred to a new glass tube. The sample was completely dried using the miVac Duo LV (Gen‐evac). The extracted lipids were dissolved in 30 μL of methanol, and 10 μL of the sample was used for lipid analysis.

Muraki R., Morita Y., Ida S., Kitajima R., Furuhashi S., Takeda M., Kikuchi H., Hiramatsu Y., Takanashi Y., Hamaya Y., Sugimoto K., Ito J., Kawata K., Kawasaki H., Sato T., Kahyo T., Setou M, & Takeuchi H. (2023). Phosphatidylcholine in bile‐derived small extracellular vesicles as a novel biomarker of cholangiocarcinoma. Cancer Medicine, 12(12), 13007-13018.

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Production and Labeling of Recombinant CD1c

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Plasmids encoding the extracellular domains of human CD1c, and human β2-microglobulin (β2m) were separately cloned into the prokaryotic expression vector pET23d (Novagen). CD1c and β2m were subsequently produced as inclusion bodies in Escherichia coli Rosetta strain (Novagen). Inclusion bodies were thoroughly washed and fully denatured then reduced in 6 M guanidineHCl and 20 mM DTT before in vitro refolding. Refolding of CD1c/β2m complexes was performed by oxidative in vitro refolding as previously described^{56 (link)} in the presence of Phosphatidylcholine (PC) (Avanti Polar Lipids). Correctly folded proteins were purified by size-exclusion chromatography using preparatory grade SD75 26/60 and analytical grade SD75 GL 10/300 gel filtration columns (GE Healthcare). Refolded CD1c-PC complexes were biotinylated via an engineered BirA motif at the C terminus, repurified by size exclusion chromatography before conjugation to dextran-PE (Immudex) to generate labelled CD1c-dextramers^{31 (link)}.

Melandri D., Zlatareva I., Chaleil R.A., Dart R.J., Chancellor A., Nussbaumer O., Polyakova O., Roberts N.A., Wesch D., Kabelitz D., Irving P.M., John S., Mansour S., Bates P.A., Vantourout P, & Hayday A.C. (2018). The γδ T cell receptor combines innate with adaptive immunity by utilizing spatially distinct regions for agonist-selection and antigen responsiveness. Nature immunology, 19(12), 1352-1365.

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Recombinant FVIII Coagulation Assay

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Recombinant FVIII (Kogenate™) was a generous gift from Dr. Lisa Regan of Bayer Corporation (Berkeley, CA). Dioleoyl phospholipids [Phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS)] were purchased from Avanti Polar Lipids (Alabaster, AL). The reagents α-thrombin, FVIIa, FIXaβ, FX, and FXa (Enzyme Research Laboratories, South Bend, IN), hirudin (DiaPharma, West Chester, OH), the chromogenic FXa substrate, Pefachrome Xa (Pefa-5523, CH₃OCO-D-CHA-Gly-Arg-pNA·AcOH; Centerchem Inc. Norwalk CT), Enhanced Chemifluorescence reagent (GE Healthcare Bioscience, Piscataway, NJ), recombinant human tissue factor (rTF: Innovin, Dade Behring, Deerfield, IL), flourogenic substrate (Z-Gly-Gly-Arg-AMC: Calbiochem, San Diego, CA), and thrombin calibrator (Diagnostica Stago, Parsippany, NJ) were purchased from the indicated vendors.

Wakabayashi H., Wintermute J.M, & Fay P.J. (2014). Combining mutations that modulate inter-subunit interactions and proteolytic inactivation enhance the stability of factor VIIIa. Thrombosis and haemostasis, 112(1), 43-52.

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Authentic Internal Standards for Lipidomic Analysis

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The authentic internal standards (IS) for lipidomic analysis, namely, phosphatidylcholine (PC) 13:0/13:0, phosphatidylethanolamine (PE) 15:0/15:0, phosphatidylinositol (PI) 8:0/8:0, lysophosphatidylcholine (LPC) 15:0, lysophosphatidylethanolamine (LPE) 13:0, and lysophosphatidylinositol (LPI) 13:0, were purchased from Avanti Polar Lipids (Alabaster, AL), while triacylglycerol (TG) 11:0/11:0/11:0 and free fatty acid (FFA) 17:0 were obtained from Sigma-Aldrich (St. Louis, MO). Unless specified, other chemicals and reagents were of the highest grade available and purchased from Sigma-Aldrich.

Chen Z., Sai S., Nagumo K., Wu Y., Chiba H, & Hui S.P. (2023). Distinctive serum lipidomic profile of IVIG-resistant Kawasaki disease children before and after treatment. PLOS ONE, 18(3), e0283710.

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Rosmarinic Acid Conjugated Lipid Synthesis

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Rosmarinic acid, dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), organic solvents, such as chloroform, dichloromethane, acetic acid and methanol, were purchased from Sigma-Aldrich Inc (MO, USA). 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine and phosphatidylcholine (PC) were bought from Avanti Polar Lipids Inc (AL, USA). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine with conjugated methoxyl poly (ethylene glycol) (DSPE-mPEG₂₀₀₀) was bought from Laysan Bio Inc (AL, USA). DOXIL was purchased from Janssen Pharmaceutica (NV, USA). LysoTracker Deep Red and Hoechst 33342 were purchased from Thermo Fisher Scientific Inc (MA, USA). The rosmarinic-lipids were characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (Bruker UltraFlextreme) and nuclear magnetic resonance spectrometer (600 MHz Bruker Avance III). The cell uptake, ROS production and apoptosis were carried on by 20-color flow cytometry (Fortessa, BD). The cellular distribution was conducted by a confocal laser scanning microscope (LSM810, Carl Zeiss). The animal imaging studies were investigated by ChemiDoc Imaging Systems (ChemiDoc XRS+, Bio-Rad). The UV-vis absorption of doxorubicin was measured by a UV-vis spectrometer (UV-1800, Shimadzu) and the fluorescence was tested by a fluorophotometer (RF-6000, Shimadzu).

Xue X., Ricci M., Qu H., Lindstrom A., Zhang D., Wu H., Lin T.Y, & Li Y. (2020). Iron-Crosslinked Rososome with Robust Stability and High Drug Loading for Synergistic Cancer Therapy. Journal of controlled release : official journal of the Controlled Release Society, 329, 794-804.

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Coagulation Factor Binding Assay

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Human FXa, thrombin, AT, and bovine lactadherin were purchased from Hematologic Technologies (Essex Junction, VT). FXa chromogenic substrate (equal parts of Bz-Ile-Glu(OCH₃)-Gly-Arg-pNA.HCl and Bz-Ile-Glu(OH)Gly-Arg-pNA.HCl) was from Aniara Diagnostica (West Chester, OH) and thrombin chromogenic substrate (H-D-Phe-Pip-Arg-pNA.2HCl) from Molecular Innovations (Novi, MI). Rabbit SkM, unfractionated heparin (UFH), low molecular weight heparin (LMWH, enoxaparin), N-acetyl-de-O-sulfated heparin (NAc-deO-Hep), and fatty acid-free and protease-free bovine serum albumin (BSA) were from Sigma Aldrich (St. Louis, MO). Bovine CM was from Cytoskeleton, Inc. (Denver, CO), heparin pentasaccharide (H5, fondaparinux) from Fisher Scientific (Hampton, NH), heparan sulfate (HS) and chondroitin sulfate (CS) from MedChemExpress (Princeton, NJ). Phosphatidylserine (PS) and phosphatidylcholine (PC) were purchased from Avanti Polar Lipids (Alabaster, AL), and recombinant human annexin V from Biovision (Milpitas, CA).

Morla S., Deguchi H, & Griffin J.H. (2020). Skeletal muscle myosin and cardiac myosin attenuate heparin’s antithrombin-dependent anticoagulant activity. Journal of thrombosis and haemostasis : JTH, 19(2), 470-477.

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Lipid Standards for Analytical Assays

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Triacylglycerol (TAG, # T5141), diacylglycerol (DAG, #D0138), monoacylglycerol (MAG, #M2015) and fatty acid (FA, #M3128) were purchased from Sigma-Aldrich. Glucosylceramide (#860539), phosphatidylcholine (PC, #850475), sphingomyelin (SM, 860584), phosphatidylinositol (PI, 840042) and lyso-PC (#845875) were purchased from Avanti polar lipids.

Wun K.S., Reijneveld J.F., Cheng T.Y., Ladell K., Uldrich A.P., Le Nours J., Miners K.L., McLaren J.E., Grant E.J., Haigh O.L., Watkins T.S., Suliman S., Iwany S., Jimenez J., Calderon R., Tamara K.L., Leon S.R., Murray M.B., Mayfield J.A., Altman J.D., Purcell A.W., Miles J.J., Godfrey D.I., Gras S., Price D.A., Van Rhijn I., Moody D.B, & Rossjohn J. (2018). T cell autoreactivity directed toward CD1c itself rather than toward carried self lipids. Nature immunology, 19(4), 397-406.

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Quantitative Lipidomics by LC-MS

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Methanol, chloroform and acetonitrile for LC-MS were purchased from Kanto Chemical (Tokyo, Japan). Ammonium formate (1 mol L-1) and formic acid for LC-MS were purchased from Wako Pure Chemical Industries (Osaka, Japan). Chemical standard phosphatidylcholine (PC) was purchased from Avanti Polar Lipids (Alabaster, AL). Chemical standards including spermine, carnitine and α-cyano-4-hydroxycinnamic acid (CHCA), were purchased from Sigma-Aldrich (Tokyo), and 9-Aminoacridine (9-AA) was purchased from Merck Schuchardt (Hohenbrunn, Germany). All chemical standards were obtained from common commercial sources.

Sato K., Saigusa D., Saito R., Fujioka A., Nakagawa Y., Nishiguchi K.M., Kokubun T., Motoike I.N., Maruyama K., Omodaka K., Shiga Y., Uruno A., Koshiba S., Yamamoto M, & Nakazawa T. (2018). Metabolomic changes in the mouse retina after optic nerve injury. Scientific Reports, 8, 11930.

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Lipid extraction and sourcing

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LPC and phosphatidylcholine (PC) extracted from chicken eggs were purchased from Avanti Polar Lipids (Alabaster, AL). Sphingosylphosphorylcholine (SPC) was from Biomol Research Laboratories (Plymouth Meeting, PA). Glycerophosphorylcholine (cadmium free) was from Kanto Kagaku (Tokyo, Japan). Other chemicals were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Morita J., Kano K., Kato K., Takita H., Sakagami H., Yamamoto Y., Mihara E., Ueda H., Sato T., Tokuyama H., Arai H., Asou H., Takagi J., Ishitani R., Nishimasu H., Nureki O, & Aoki J. (2016). Structure and biological function of ENPP6, a choline-specific glycerophosphodiester-phosphodiesterase. Scientific Reports, 6, 20995.

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Recombinant Ribonuclease Production and Assays

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Murine ribonuclease 1 (RNase 1) and ribonuclease A were produced in E. coli as described previously (delCardayré et al. 1995 (link); Lomax et al. 2014 (link)). PNGase F was from New England BioLabs. Human FXI-, FXII-, and PK-deficient plasma was from George King Bio-Medical. Murine FXI- and FXII-deficient plasma (Gailani et al. 1997 (link); Pauer et al. 2004 (link)), and FIX-deficient plasma (Lin et al. 1997 (link)) were obtained as described. Soluble tissue factor was a gift from B.S. Schwartz (University of Wisconsin–Madison).
Phosphatidylserine (PS), phosphatidylcholine (PC), and cholesterol were from Avanti Polar Lipids. PC:PS:C (molar ratio, 75:25:20) phospholipid vesicles were prepared by extrusion through a 100-nm polycarbonate filter. Primers, DNA constructs, and the 6-FAM–dArUdAdA–6-TAMRA ribonuclease substrate were from Integrated DNA Technologies. Poly(cytidylic acid) and LPS were from Sigma–Aldrich.

Garnett E.R., Lomax J.E., Mohammed B.M., Gailani D., Sheehan J.P, & Raines R.T. (2019). Phenotype of ribonuclease 1 deficiency in mice. RNA, 25(8), 921-934.

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