3’,6-dinonylneamine was synthesized by Decout and colleagues [21 ;22 (link)]. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,3-bis-(sn-3’-phosphatidyl)-sn-glycerol (cardiolipin; CL: from E. coli) were purchased from Avanti Polar Lipids (Alabaster, AL). Octadecyl rhodamine B (R18) was purchased from Invitrogen (Paisley, Scotland, UK). Laurdan and calcein were purchased from Sigma-Aldrich. All solvents (analytical grade) were purchased from E. Merck AG.
Octadecyl rhodamine b r18
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Octadecyl rhodamine B (R18) is a fluorescent dye that is commonly used in scientific research. It is a lipophilic dye that can be used to label and track lipid-rich structures and membranes within cells. The dye's core function is to provide a fluorescent signal that allows for the visualization and analysis of these cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Calcein, by Merck Group (2 mentions)
Laurdan, by Merck Group (2 mentions)
1 palmitoyl 2 oleoyl sn glycero 3 phosphoglycerol, by Avanti Polar Lipids (2 mentions)
1 palmitoyl 2 oleoyl sn glycero 3 phosphoethanolamine, by Avanti Polar Lipids (1 mentions)
Complete protease inhibitory tablets, by Roche (1 mentions)
Calcein, by Thermo Fisher Scientific (1 mentions)
Pdms sylgard 184, by Dow (1 mentions)
1 2 dioleoyl sn glycero 3 phospho 1 rac glycerol, by Avanti Polar Lipids (1 mentions)
Resazurin, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Staphylococcus aureus atcc33591, by American Type Culture Collection (1 mentions)
6 protocols using octadecyl rhodamine b r18
1
Synthesis and Lipid Characterization Protocol
2
Labeling and Purification of SLST A1 EVs
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EVs labelling was performed as previously described17 (link),23 (link),24 (link). Briefly, isolated SLST A1 EVs were resuspended in PBS buffer, centrifuged at 154,300 × g for 2 h at 4 °C, and resuspended in labelling buffer (50 mM Na2CO3, 100 mM NaCl, pH 9.2) in the presence of 1 mg/mL of octadecyl rhodamine B-R18 (Invitrogen) and incubated for 1 h at 25 °C. Labelled SLST A1 EVs were pelleted by ultracentrifugation at 154,300 × g for 1 h at 4 °C, resuspended in PBS buffer (0.2 M NaCl) and washed three times to fully remove the unbound dye. After the final ultracentrifugation step, B-R18 labelled A1 EVs were resuspended in PBS buffer (0.2 M NaCl) containing a protease inhibitory cocktail (Complete Protease Inhibitory Tablets, Roche) and were stored at − 20 °C.
3
Lipid Bilayer Formation and Characterization
4
Antimicrobial Evaluation of Ursolic and Oleanolic Acids
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Ursolic acid (UA) and oleanolic acid (OA) (purity 98%) were obtained from Ava Chem (San Antonio, TX, USA) or Extrasynhese (Genay, France). 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG; Tm: −2 °C), 1,3-bis-(sn-3′-phosphatidyl)-sn-glycerol (cardiolipin; CL from E. coli) and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))] (chloride salt) (C18:1 lysyl-phosphatidylglycerol) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Octadecyl rhodamine B (R18) was purchased from Invitrogen (Paisley, Scotland, UK). Laurdan, calcein, DMSO and resazurin were ordered to Sigma, St. Louis, MO, USA. All solvents (analytical grade) were purchased from E. Merck AG. Tryptic soy agar (TSA) was bought to Difco (Difco, Richmond, CA, USA). High-performance thin-layer chromatography (HPTLC) plates precoated with silica gel 60 F254 were purchased from Merck KGaA (Darmstadt, Germany).
Staphylococcus aureus ATCC33591 (MRSA and β-lactamase producer; American Type Culture Collection, Manassas, VA, USA) and COL (HA-MRSA SCCmec type 1 strain COL) [19 (link),20 (link)];http://www.tigr.org/tdb/staphylococcus (accessed on 10 January 2021) were used.
Staphylococcus aureus ATCC33591 (MRSA and β-lactamase producer; American Type Culture Collection, Manassas, VA, USA) and COL (HA-MRSA SCCmec type 1 strain COL) [19 (link),20 (link)];
5
Viral Envelope Labeling with R18 Dye
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To label the viral envelope with lipophilic fluorescent dye, 5 μL of the virus solution, 250 μL of MES buffer, and 4 μL of 0.01 mg/mL ethanol-dissolved octadecyl rhodamine B (R18) [Invitrogen, Carlsbad, CA] were mixed together in a vial. The mixed solution was gently sonicated in a water bath for 30 min at 25°C in the dark. Unincorporated R18 dye was filtered out using a G-25 sephadex spin column [GE Healthcare, Pittsburgh, PA] at 3000 RPM (743 RCF), and the eluted virus solution was stored in a LoBind vial [Eppendorf, Hamburg, Germany] to prevent loss of viral particles to vial surfaces while conducting the experiments. Before use, 250 μL of filtered virus solution was diluted with 1 mL of MES buffer. Note the labeling of virus with membrane dyes has already been shown to not affect HA function [52 (link)–53 (link)].
6
Hemifusion Assay for Viral Glycoproteins
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The lipid probe octadecyl rhodamine B (R18) (Invitrogen, California, USA) was used to determine the ability of HN mutations to promote hemifusion from RBCs to BHK-21 cells co-transfected with HN and F genes as the protocol previously described (29) . RBCs were washed and re-suspended with PBS, then labeled with R18 (1 mg/mL in ethanol) at room temperature for 30 min in the dark. RBCs were washed 5 times with ice-cold PBS to remove the uncombined R18 and re-suspended in PBS (0.1% hematocrit). Labeled RBCs were added to co-transfected monolayers and incubated at 4°C for 30 min. Cells were washed with PBS to remove unbonded RBCs, then incubated and the fluorescence was visualized using a fluorescence microscope (OLYMPUS, Japan) at 37°C.
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