Lipid rafts were isolated as previously described by discontinuous sucrose gradients ultracentrifugation [28 (link),29 (link),64 (link)]. Briefly, 70 ± 5 mg of brain cortical tissue was homogenized, the nuclear fraction removed, and the cell membrane pellet obtained by centrifugation (100,000× g at +4 °C, 30 min; ultracentrifuge Beckman Optima XL-80 K, Beckman Coulter, Inc., Brea, CA, USA). The pellet was homogenized in a buffer containing the non-ionic detergent Brij O20 (Sigma-Aldrich, St. Louis, MO, USA) and ultracentrifuged (140,000× g, at +4 °C, 18 h) in a discontinuous sucrose (Kemika, Zagreb, Croatia) gradient which consisted of 85% sucrose mixed with sample in 1:1 ratio, overlaid with 35% sucrose solution followed by 3% sucrose solution (ultracentrifuge Beckman Optima XL-80 K, Beckman Coulter, Inc., Brea, CA, USA). After centrifugation, fractions were collected top to bottom and analyzed by Western blotting.
Brij o20
Manufactured by Merck Group
Sourced in United States, United Kingdom
Brij O20 is a non-ionic surfactant. It is a polyoxyethylene (20) oleyl ether used as a wetting and emulsifying agent in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Brij o10, by Merck Group (2 mentions)
Chloroform d cdcl3, by Cambridge Isotopes (2 mentions)
Brij l23, by Merck Group (2 mentions)
Beckman optima xl 80 k, by Beckman Coulter (1 mentions)
Sodium hydroxide (naoh), by Chempur (1 mentions)
Tween 20, by Avantor (1 mentions)
Brij c10, by Merck Group (1 mentions)
Genapol x 080, by Merck Group (1 mentions)
Tween 80, by Avantor (1 mentions)
Brij 35, by Merck Group (1 mentions)
Sodium deoxycholate doc, by Merck Group (1 mentions)
Polyethylene glycol 200 peg 200, by Thermo Fisher Scientific (1 mentions)
Absolute ethanol etoh, by Thermo Fisher Scientific (1 mentions)
Dulbecco a, by Thermo Fisher Scientific (1 mentions)
Hplc grade water, by Thermo Fisher Scientific (1 mentions)
Methanol, by Merck Group (1 mentions)
Sodium carbonate, by Thermo Fisher Scientific (1 mentions)
Dichloromethane, by Thermo Fisher Scientific (1 mentions)
Trifluoroacetic acid, by Thermo Fisher Scientific (1 mentions)
Magnesium sulfate, by Thermo Fisher Scientific (1 mentions)
11 protocols using brij o20
1
Isolation of Lipid Rafts from Brain Tissue
2
Transdermal Diclofenac Gel Formulation
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IBU, methanol, Brij® O20, or Polyoxyethylene (20) oleyl ether were provided by Sigma-Aldrich (Dorset, UK). All other materials were of analytical grade unless otherwise specified. The formulations tested, Nurofen Joint & Back Pain Relief Max Strength 10% Gel (B/N: P607; Mercury Pharma Group Ltd., London, UK) and Ibuleve Speed Relief Max Strength 10% Gel (B/N: X1032; Diomed Developments Ltd., Herts, UK), were purchased from a local pharmacy. Excised abdominal human skin from one single donor was obtained from cosmetic surgery procedures with informed consent and institutional ethical approval (Research Ethics Committee reference 07/H1306/98). Human tissues were preprepared as described in a previous paper [19 (link)].
3
Aqueous Two-Phase Extraction (ATPE) of SWCNTs
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Dextran (DEX, MR ≈70 kDa, PanRecAppliChem, Germany), poly(ethylene glycol) (PEG, Mn ≈6000 Da, Alfa Aesar, Germany), sodium cholate (SC, PanRecAppliChem, Germany), sodium deoxycholate (DOC, Sigma‐Aldrich, USA), Triton X100 (TX100, Thermo Fisher Scientific, USA), Brij 35 (Sigma‐Aldrich, USA), Tween‐20 (VWR, USA), Brij O20 (Sigma‐Aldrich, USA), Brij O10 (Sigma‐Aldrich, USA), Genapol X080 (Sigma‐Aldrich, USA), Brij C10 (Sigma‐Aldrich, USA), Tween‐80 (VWR, USA), and sodium hydroxide (Chempur, Poland) were all of pure p.a. class, so they were employed without any further purification. For this work, Signis SG65i SWCNTs (Sigma‐Aldrich, LOT: MKCK1004) enriched in (6,5) chirality to simplify the interpretation of SWCNT migration between the phases due to the reduced number of species. Wherever specified, HiPco (Nanointegris, Canada; LOT: HP30‐006, purified) and Signis SG76i (Sigma‐Aldrich, LOT: MKBZ1157V) SWCNTs as references were also used to investigate the ATPE mechanics. For all sorting experiments and characterization, double‐distilled water obtained from the Elix Millipore system was used.
4
Topical Formulation of Terbinafine HCl
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TBF hydrochloride was donated by GSK Ltd. (Weybridge, UK), while TBF-free base was synthesised, purified, and characterised in house. Dichloromethane, sodium carbonate, magnesium sulfate, polyethylene glycol 200 (PEG 200), HPLC grade water, isopropyl alcohol (IPA), and absolute ethanol (EtOH) were supplied by Fisher Scientific (Leicestershire, UK). HPLC grade solvents, including methanol and polyoxyethylene (20) oleyl ether (Brij® O20) were obtained from Sigma-Aldrich (Dorset, UK). PG and IPM (analytical grade) were purchased from Alfa Aesar (Thermo Fisher Scientific, Lancashire, UK). TC and PGML (analytical grade) were gifts from Gattefossé (St. Priest, France). Trifluoroacetic acid was supplied by Acros Organics (Fisher Scientific, Leicestershire, UK) and OL from Fluka (Sigma-Aldrich, Dorset, UK). Chloroform-D (CDCl3) was obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). Phosphate buffered saline (PBS) tablets (Dulbecco A, pH 7.3 ± 0.2 at 25 °C) were purchased from Oxoid Limited (Cheshire, UK), and high-vacuum grease was obtained from Dow Corning (Seneffe, Belgium). Lamisil® AT gel (TBF 1% w/w, GlaxoSmithKline, Brentford, UK) was purchased from a local pharmacy. Porcine ear skin was sourced from a local abattoir.
5
Lipid-Based siRNA Delivery Protocol
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2, 3-dioleoyloxy-propyl-trimethylammoniumchlorid (DOTAP) was purchased from CordenPharma (Switzerland). Phospholipon 90G was purchased from Lipoid GmbH (Germany). Thiazolyl Blue Tetrazolium Bromide (MTT) and BRIJ O20 were purchased from Sigma-Aldrich (St. Louis Missouri, USA). FAM-labeled GAPDH-siRNA (5′-FAM-CACUCAAGAUUGUCAGCAATT-3′) was obtained from GenePharma (Shanghai, China). Neutral Balsam was purchased from Solarbio (Beijng, China). L929 cell was purchased from iCell Bioscience (Shanghai, China). Trypsin 0.25% (1× ) Solution, Dulbecco’s modified Eagle’s medium (DMEM)/high glucose, and 100× penicillin/streptomycin solution were purchased from HyClone (Logan City, UT, USA). Fetal bovine serum (FBS) and opti-mem (1×) were purchased from GIBCO (Grand Island, USA). Chloral hydrate was purchased from Macklin (Shanghai, China). KDalert GAPDH Assay was purchased from Thermo Scientific (USA). Female BALB/c mice (6–7 weeks old) were purchased from Xiamen University Laboratory Animal Center. All other chemicals (analytical grade) used in this study were purchased from Sinopharm Group (Shanghai, China).
6
Transdermal Permeation Enhancement Excipients
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Methadone hydrochloride, triPropylene glycol (TriPG), diPropylene glycol (DiPG), 1-octanol, 1,3-butanediol (1,3-BD), Brij™ O20, isopropyl myristate (IPM), octyl salicylate (OSAL), oleic acid (OA) and ethyl oleate (EO) were purchased from Sigma-Aldrich, Dorset, UK. Propylene glycol (PG), ethyl acetate, d-limonene (LIM), high performance liquid chromatography (HPLC) grade water, acetonitrile, orthophosphoric acid, 85+%, sodium dihydrogen phosphate, sodium hydrogen phosphate and sodium hydroxide were supplied by Fisher Scientific, Loughborough, UK. Chloroform-d (CDCl3) was purchased from Cambridge Isotope Laboratories Tewksbury, MA, USA. Labrafac™ lipophile WL 1349 (medium-chain triglycerides of caprylic and capric acids, WL 1349), Transcutol® (TC), Propylene glycol monocaprylate (PGMC) Type II and Propylene glycol monolaurate (PGML) Type II were gifts from Gattefossé, St. Priest, France. Phosphate buffered saline (PBS) (pH 7.3 ± 0.2 at 25 °C) was prepared using Dulbecco A tablets supplied by Oxoid, Cheshire, UK. Full thickness porcine ear skin was obtained from a local abattoir.
7
Evaluation of P-glycoprotein Modulators
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BBR was purchased from Oddfoni Biological Technology Company (Nanjing, China). Verapamil, cyclosporin A (CsA), and Rhodamine123 were purchased from J&K Scientific Ltd. (Bei-jing, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), methanol, dimethyl sulphoxide (DMSO), and Brijs (Brij-L4, decaethylene glycol monododecyl ether [BD], Brij-L23, Brij-52 Brij-C10, Brij-58, Brij-93, Brij-O10, Brij-O20, Brij-S2, Brij-S10, Brij-S20, Brij-S100, D-α-Tocopherol polyethylene glycol 1000 succinate [TPGS]) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). High-performance liquid chromatography (HPLC) grade acetonitrile was purchased from Merck Company (Darmstadt, Germany).
ATPlite luminescence ATP detection assay system was bought from Perkin Elmer (Waltham, MA, USA). Pgp-Glo assay system with human Pgp membrane was obtained from Promega (Madison, WI, USA). All materials for cell culture were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies against Pgp and GAPDH, and anti-rabbit IgG were obtained from Cell Signaling Technology (Beverly, MA, USA).
ATPlite luminescence ATP detection assay system was bought from Perkin Elmer (Waltham, MA, USA). Pgp-Glo assay system with human Pgp membrane was obtained from Promega (Madison, WI, USA). All materials for cell culture were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies against Pgp and GAPDH, and anti-rabbit IgG were obtained from Cell Signaling Technology (Beverly, MA, USA).
8
Modeling Active Ingredient Release Kinetics
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Active ingredients release kinetics was modelled with two different dyes–hydrophilic Rhodamine 6G and hydrophobic Nile red. As a polar environment for Rhodamine release, distilled water was selected. For hydrophobic eluate release, water solution of polyoxyethylene (20) oleyl ether (Brij®O20, Sigma Aldrich, St. Louis, MO, USA) was used as the solubilizing agent. Dye was added to polymer solution to consequential concentration of 1 g/L. Subsequently, films were prepared as described above. Pieces of 2.6 × 2.6 cm-coloured films were incubated in solvent for 48 h. Amount of released dye was determined spectrometrically at 524 nm for Rhodamine and 561 nm for Nile red detection.
9
Formulation of Drug-Loaded Nano-Carriers
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Materials were obtained from Sigma unless otherwise noted. The following were used: Pluronic F127 (Sigma # P2443), Pluronic F108 (Sigma # 542342), Pluronic F68 (Sigma # 412325), Polysorbate 20 (Amresco # M147-1L), Polysorbate 40 (Sigma # 1547936), Polysorbate 80 (VWR # EM-9490), Cremophor EL (Sigma # C5135), Cremophor RH40 (Sigma # 07076), Tergitol NP 9 (Sigma # NP9), Tergitol NP 10 (Sigma # NP10), Tergitol NP 40 (Sigma # NP40S), Brij 97 (Spectrum # B1679), Brij 35 (Spectrum # BR105), Brij L23 (Sigma # 16005), Brij O20 (Sigma # 436240), Vitamin K1 (VK1, VWR # AAAL10575-03), cyclosporine A (CsA, VWR # 89156-334), Testosterone undecanote (T-undec, Matrix # 099258), cholecalciferol (VWR, TCC0314), Retinol Palmitate (VWR # IC15652125), coenzyme Q10 (Sigma # 45-C9538), Docetaxel (LC Labs # D-1000), Paclitaxel (LC Labs # P-9600), Cabazitaxel (CTX, Proactive Molecular Research), Squalene (VWR # AAB20944-22), 5,9,14,18,23,27,32,36-Octabutoxy-2,3-naphthalocyanine (ONc, Sigma # 412074) 2,9,16,23-Tetra-tert-butyl-29H,31H-phthalocyanine (BPc, Sigma # 423157), Zinc 2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine (ZnBNc, Sigma # 432210), Castor oil (Sigma # 259853), methylene chloride (Fisher), Ethanol (Decon), ethylene glycol (VWR # 1071401), and sodium chloride (Macron # 7532-06).
10
Protein Analysis via Spectroscopy
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Sodium chloride (Sigma, S7653), Brij O-20 (polyoxyethylene (20) oleyl ether, Sigma, 436240,), Ex-CELL 610-HSF medium (Sigma, 14610C), glycine (Bio-lab 07132391), bathophenanthroline (GFS chemicals, C038446), ferrous chloride tetrahydrate (Sigma, 44939), human IgG (Lee-Biosciences, 340-21), phenylalanine (Sigma, P2126), tyrosine (Sigma, T3754), tryptophan (Sigma, T3300), isoleucine (Sigma, I2752), valine (Sigma, 94640), Polyethylene glycol-6000 (PEG-6000) (Sigma, 81260).
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