triethylammonium salt  (Thermo Fisher)


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    Structured Review

    Thermo Fisher triethylammonium salt
    Triethylammonium Salt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/triethylammonium salt/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    triethylammonium salt - by Bioz Stars, 2020-02
    99/100 stars

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    Related Articles

    High Performance Liquid Chromatography:

    Article Title: Mononuclear phagocyte intercellular crosstalk facilitates transmission of cell-targeted nanoformulated antiretroviral drugs to human brain endothelial cells
    Article Snippet: Labeling of nanoART NanoART were fluorescently labeled using lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (excitation 560 nm, emission 580 nm), or the Vybrant 3,3′-dioctadecyloxacarbo-cyanine perchlorate (DiD) cell-labeling solution (excitation 644 nm: emission 665 nm) (Invitrogen, Carlsbad, CA). .. NanoART particles were then washed at least five times with phosphate buffered saline (PBS) to remove all excess dye, and final drug concentration of the formulations was determined by HPLC.

    Article Title: Monitoring Phases and Phase Transitions in Phosphatidylethanolamine Monolayers Using Active Interfacial Microrheology
    Article Snippet: .. Texas Red® 1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine, triethylammonium salt, (TXR-DHPE) was purchased in the dried form from Life Technologies (Invitrogen) and dissolved in HPLC grade chloroform. .. All organic solvents were purchased from Fisher Scientific.

    Article Title: Phospholipid Composition Modulates Carbon Nanodiamond-Induced Alterations in Phospholipid Domain Formation
    Article Snippet: .. Texas red 1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine, triethylammonium salt, (TXR-DHPE) lipid dye was purchased in the dried form from Life Technologies (Invitrogen, Grand island, NY) and dissolved in high-performance liquid chromatography (HPLC)-grade chloroform (final concentration 0.5 mg/mL). .. All organic solvents used for this work were purchased from Thermo Fisher Scientific Inc. (Pittsburgh, PA).

    Article Title: Bilayer Asymmetry Influences Integrin Sequestering in Raft-Mimicking Lipid Mixtures
    Article Snippet: The dye-labeled lipids NBD-DHPE ( n -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadec-anoyl- sn -glycero-3-phosphoethanolamine), TRITC-DHPE ( n -(6-tetramethylrhodamine-thiocarbamoyl)-1, 2-dihexadecanayl- sn -glycero-3-phosphoethanolamine, triethylammonium salt), DID (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate salt), and DiI (1,11 -dioctacadecayl-3,3,31 ,31 -tetramethylindocarbocyanine perchlorate), as well as the kits for fluorescently labeling antibodies with Alexa-555 were obtained from Invitrogen (Carlsbad, CA). .. Chloroform (HPLC grade; Fisher Scientific, Pittsburgh, PA) was used as the spreading solvent for the lipid monolayer at the air-water interface in the trough.

    Article Title: Transition from Nanodomains to Microdomains Induced by Exposure of Lipid Monolayers to Air
    Article Snippet: High-performance liquid chromatography grade chloroform and methanol were from ACP Chemicals (Quebec, Canada). .. Texas Red 1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red DHPE) was purchased from Invitrogen Canada (Ontario, Canada).

    Flow Cytometry:

    Article Title: DNA compaction by the bacteriophage protein Cox studied on the single DNA molecule level using nanofluidic channels
    Article Snippet: The sample is introduced into the channel system via one of the four reservoirs that are connected to the microchannels and moved inside the channels by flow induced by applying pressure on the reservoir inlets of the microchannels. .. To avoid non-specific binding of protein to the negatively charged channels walls, the channels were prior to sample introduction coated with a lipid bilayer comprising 99% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, Avanti) and 1% lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt, (rhodamine-DHPE, Invitrogen).

    Luciferase:

    Article Title: Patterned Thread-like Micelles and DNA-Tethered Nanoparticles: A Structural Study of PEGylated Cationic Liposome–DNA Assemblies
    Article Snippet: For fluorescence experiments liposomes were prepared with 0.2 wt% Texas Red® - 1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red® DHPE, excitation/emission 595/615 nm) from Invitrogen (Carlsbad, CA). .. Four distinct types of DNA were used to form nanoparticles; UltraPure Salmon Sperm DNA Solution (S-DNA) (Invitrogen (Carlsbad, CA)), Lambda Phage DNA (λ-DNA) (Thermo Scientific (Waltham, MA)), pGL3 Luciferase Reporter plasmid DNA (pGL3) (Promega (Fitchburg, Wisconsin)), which was propagated via Qiagen Plasmid Plus Mega Kit (Venlo, Limburg) and 11 bp DNA (purchased as single strands from Sigma-Genosys (Sigma-Aldrich (St. Louis, MI) and delivered as a lyophilized film).

    Binding Assay:

    Article Title: DNA compaction by the bacteriophage protein Cox studied on the single DNA molecule level using nanofluidic channels
    Article Snippet: .. To avoid non-specific binding of protein to the negatively charged channels walls, the channels were prior to sample introduction coated with a lipid bilayer comprising 99% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, Avanti) and 1% lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt, (rhodamine-DHPE, Invitrogen). ..

    Fluorescence:

    Article Title: Patterned Thread-like Micelles and DNA-Tethered Nanoparticles: A Structural Study of PEGylated Cationic Liposome–DNA Assemblies
    Article Snippet: .. For fluorescence experiments liposomes were prepared with 0.2 wt% Texas Red® - 1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red® DHPE, excitation/emission 595/615 nm) from Invitrogen (Carlsbad, CA). .. Four distinct types of DNA were used to form nanoparticles; UltraPure Salmon Sperm DNA Solution (S-DNA) (Invitrogen (Carlsbad, CA)), Lambda Phage DNA (λ-DNA) (Thermo Scientific (Waltham, MA)), pGL3 Luciferase Reporter plasmid DNA (pGL3) (Promega (Fitchburg, Wisconsin)), which was propagated via Qiagen Plasmid Plus Mega Kit (Venlo, Limburg) and 11 bp DNA (purchased as single strands from Sigma-Genosys (Sigma-Aldrich (St. Louis, MI) and delivered as a lyophilized film).

    Synthesized:

    Article Title: Fabrication and characterization of spatially-defined, multiple component, chemically-functionalized domains in enclosed silica channels using cross-linked phospholipid membranes
    Article Snippet: DOPE-PEG-pNP ( p -nitrophenylcarbonyl-PEG-1,2-dioleoyl- sn -glycero-3-phosphoethanolamine) was synthesized and purified following published procedures. .. ; FM 1-43, biotin-DHPE ( N -(biotinoyl)-1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine, triethylammonium salt), streptavidin-R-phycoerythrin (SA-RPE), streptavidin-fluorescein conjugate and 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) were from Molecular Probes (Eugene, OR).

    Centrifugation:

    Article Title: Mononuclear phagocyte intercellular crosstalk facilitates transmission of cell-targeted nanoformulated antiretroviral drugs to human brain endothelial cells
    Article Snippet: Labeling of nanoART NanoART were fluorescently labeled using lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (excitation 560 nm, emission 580 nm), or the Vybrant 3,3′-dioctadecyloxacarbo-cyanine perchlorate (DiD) cell-labeling solution (excitation 644 nm: emission 665 nm) (Invitrogen, Carlsbad, CA). .. For labeling, 1 mL nanoART suspension was mixed overnight with 5 μL of dye and nanoART pelleted by centrifugation at 20,000 ×g for 5 minutes.

    Cell Culture:

    Article Title: Synthetic Protocells Interact with Viral Nanomachinery and Inactivate Pathogenic Human Virus
    Article Snippet: Red fluorescent dye, 1,2- dihexadecanoyl-sn-glycero-3- phosphoethanolamine, triethylammonium salt (Texas-red DHPE), was purchased from Invitrogen. .. Dilute phosphate buffered saline (PBS) for washing and formation of lipid vesicles was made by diluting 100 mL cell-culture grade 1X PBS with 400 mL of deionized (DI) water.

    Purification:

    Article Title: Fabrication and characterization of spatially-defined, multiple component, chemically-functionalized domains in enclosed silica channels using cross-linked phospholipid membranes
    Article Snippet: DOPE-PEG-pNP ( p -nitrophenylcarbonyl-PEG-1,2-dioleoyl- sn -glycero-3-phosphoethanolamine) was synthesized and purified following published procedures. .. ; FM 1-43, biotin-DHPE ( N -(biotinoyl)-1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine, triethylammonium salt), streptavidin-R-phycoerythrin (SA-RPE), streptavidin-fluorescein conjugate and 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) were from Molecular Probes (Eugene, OR).

    Concentration Assay:

    Article Title: Mononuclear phagocyte intercellular crosstalk facilitates transmission of cell-targeted nanoformulated antiretroviral drugs to human brain endothelial cells
    Article Snippet: Labeling of nanoART NanoART were fluorescently labeled using lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (excitation 560 nm, emission 580 nm), or the Vybrant 3,3′-dioctadecyloxacarbo-cyanine perchlorate (DiD) cell-labeling solution (excitation 644 nm: emission 665 nm) (Invitrogen, Carlsbad, CA). .. NanoART particles were then washed at least five times with phosphate buffered saline (PBS) to remove all excess dye, and final drug concentration of the formulations was determined by HPLC.

    Article Title: Phospholipid Composition Modulates Carbon Nanodiamond-Induced Alterations in Phospholipid Domain Formation
    Article Snippet: .. Texas red 1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine, triethylammonium salt, (TXR-DHPE) lipid dye was purchased in the dried form from Life Technologies (Invitrogen, Grand island, NY) and dissolved in high-performance liquid chromatography (HPLC)-grade chloroform (final concentration 0.5 mg/mL). .. All organic solvents used for this work were purchased from Thermo Fisher Scientific Inc. (Pittsburgh, PA).

    Article Title: Dynamic Measurements of Membrane Insertion Potential of Synthetic Cell Penetrating Peptides
    Article Snippet: 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG), 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3 phosphocholine (sodium salt) (POPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Avanti Polar Lipids, Alabaster, AL, as organic mixtures in chloroform at a final concentration of 5 mg/ml. .. Texas Red ® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt, (TXR-DHPE) lipid dye was purchased in the dried form from Life Technologies (Invitrogen Corp., Carlsbad, CA, USA).

    Article Title: Mechanistic principles underlying regulation of the actin cytoskeleton by phosphoinositides
    Article Snippet: Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (rhodamine DHPE), and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate were purchased from Thermo-Fisher Scientific. .. The concentration of the PI(4,5)P2 stock solution was determined based on a phosphate assay ( ).

    Article Title: Patterned Thread-like Micelles and DNA-Tethered Nanoparticles: A Structural Study of PEGylated Cationic Liposome–DNA Assemblies
    Article Snippet: For fluorescence experiments liposomes were prepared with 0.2 wt% Texas Red® - 1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red® DHPE, excitation/emission 595/615 nm) from Invitrogen (Carlsbad, CA). .. Complementary single strands were mixed at an equimolar ratio, diluted to a final concentration of 10 mg/mL, heated in a water bath and held at 90 °C for 15 min and slowly cooled to room temperature to allow complete hybridization.

    Labeling:

    Article Title: Mononuclear phagocyte intercellular crosstalk facilitates transmission of cell-targeted nanoformulated antiretroviral drugs to human brain endothelial cells
    Article Snippet: .. Labeling of nanoART NanoART were fluorescently labeled using lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (excitation 560 nm, emission 580 nm), or the Vybrant 3,3′-dioctadecyloxacarbo-cyanine perchlorate (DiD) cell-labeling solution (excitation 644 nm: emission 665 nm) (Invitrogen, Carlsbad, CA). .. For labeling, 1 mL nanoART suspension was mixed overnight with 5 μL of dye and nanoART pelleted by centrifugation at 20,000 ×g for 5 minutes.

    Article Title: Bilayer Asymmetry Influences Integrin Sequestering in Raft-Mimicking Lipid Mixtures
    Article Snippet: .. The dye-labeled lipids NBD-DHPE ( n -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadec-anoyl- sn -glycero-3-phosphoethanolamine), TRITC-DHPE ( n -(6-tetramethylrhodamine-thiocarbamoyl)-1, 2-dihexadecanayl- sn -glycero-3-phosphoethanolamine, triethylammonium salt), DID (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate salt), and DiI (1,11 -dioctacadecayl-3,3,31 ,31 -tetramethylindocarbocyanine perchlorate), as well as the kits for fluorescently labeling antibodies with Alexa-555 were obtained from Invitrogen (Carlsbad, CA). .. Chloroform (HPLC grade; Fisher Scientific, Pittsburgh, PA) was used as the spreading solvent for the lipid monolayer at the air-water interface in the trough.

    Staining:

    Article Title: DNA compaction by the bacteriophage protein Cox studied on the single DNA molecule level using nanofluidic channels
    Article Snippet: To avoid non-specific binding of protein to the negatively charged channels walls, the channels were prior to sample introduction coated with a lipid bilayer comprising 99% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, Avanti) and 1% lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt, (rhodamine-DHPE, Invitrogen). .. DNA from phage lambda (λ-DNA, Roche) was pre-stained with YOYO-1 (YOYO, Invitrogen) at a ratio of 1 dye molecule per 10 basepairs by mixing at high ionic strength (5× TBE, 445 mM Tris-Borate pH 8.3, 10 mM EDTA, prepared from 10× TBE tablets, Medicago) and incubating at 50°C for 1.5 h to achieve high homogeneity in the staining of the DNA ( ).

    Modification:

    Article Title: Fabrication and characterization of spatially-defined, multiple component, chemically-functionalized domains in enclosed silica channels using cross-linked phospholipid membranes
    Article Snippet: Bis-SorbPC was synthesized by a modification of the procedure reported by Lamparski et al. ; DOPC (1,2-dioleoyl- sn -glycero-3-phosphocholine), maleimide lipid (1,2-distearoyl- sn -glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol) 2000] (ammonium salt)), biotin lipid (1,2-dipalmitoyl- sn -glycero-3-phosphoethanolamine-N-(Cap Biotinyl), Rh-DPPE (1,2-dipalmitoyl- sn -glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt)) and NBD-DOPE (1,2-dioleoyl- sn -glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt)) were from Avanti Polar Lipids (Alabaster, AL). .. ; FM 1-43, biotin-DHPE ( N -(biotinoyl)-1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine, triethylammonium salt), streptavidin-R-phycoerythrin (SA-RPE), streptavidin-fluorescein conjugate and 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) were from Molecular Probes (Eugene, OR).

    Sonication:

    Article Title: Bilayer Asymmetry Influences Integrin Sequestering in Raft-Mimicking Lipid Mixtures
    Article Snippet: The dye-labeled lipids NBD-DHPE ( n -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadec-anoyl- sn -glycero-3-phosphoethanolamine), TRITC-DHPE ( n -(6-tetramethylrhodamine-thiocarbamoyl)-1, 2-dihexadecanayl- sn -glycero-3-phosphoethanolamine, triethylammonium salt), DID (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate salt), and DiI (1,11 -dioctacadecayl-3,3,31 ,31 -tetramethylindocarbocyanine perchlorate), as well as the kits for fluorescently labeling antibodies with Alexa-555 were obtained from Invitrogen (Carlsbad, CA). .. Glass coverslips were prepared by first baking them for 3 h at 515°C in a kiln followed by subsequent sonication steps in a bath sonicator using solutions of 1% sodium dodecyl sulfate for 45 min, MeOH saturated with NaOH, and 0.1% HCl (Fisher Scientific).

    Evaporation:

    Article Title: Phospholipid Composition Modulates Carbon Nanodiamond-Induced Alterations in Phospholipid Domain Formation
    Article Snippet: Texas red 1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine, triethylammonium salt, (TXR-DHPE) lipid dye was purchased in the dried form from Life Technologies (Invitrogen, Grand island, NY) and dissolved in high-performance liquid chromatography (HPLC)-grade chloroform (final concentration 0.5 mg/mL). .. The lipid mixtures were stored at −20 °C when not in use to ensure no evaporation of the organic solvent.

    Plasmid Preparation:

    Article Title: Patterned Thread-like Micelles and DNA-Tethered Nanoparticles: A Structural Study of PEGylated Cationic Liposome–DNA Assemblies
    Article Snippet: For fluorescence experiments liposomes were prepared with 0.2 wt% Texas Red® - 1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red® DHPE, excitation/emission 595/615 nm) from Invitrogen (Carlsbad, CA). .. Four distinct types of DNA were used to form nanoparticles; UltraPure Salmon Sperm DNA Solution (S-DNA) (Invitrogen (Carlsbad, CA)), Lambda Phage DNA (λ-DNA) (Thermo Scientific (Waltham, MA)), pGL3 Luciferase Reporter plasmid DNA (pGL3) (Promega (Fitchburg, Wisconsin)), which was propagated via Qiagen Plasmid Plus Mega Kit (Venlo, Limburg) and 11 bp DNA (purchased as single strands from Sigma-Genosys (Sigma-Aldrich (St. Louis, MI) and delivered as a lyophilized film).

    Hybridization:

    Article Title: Patterned Thread-like Micelles and DNA-Tethered Nanoparticles: A Structural Study of PEGylated Cationic Liposome–DNA Assemblies
    Article Snippet: For fluorescence experiments liposomes were prepared with 0.2 wt% Texas Red® - 1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red® DHPE, excitation/emission 595/615 nm) from Invitrogen (Carlsbad, CA). .. Complementary single strands were mixed at an equimolar ratio, diluted to a final concentration of 10 mg/mL, heated in a water bath and held at 90 °C for 15 min and slowly cooled to room temperature to allow complete hybridization.

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    Thermo Fisher alexafluor 594
    localisation of FLAG tagged PATs and PPTs in  Trypanosoma cruzi  epimastigotes by immunofluorescence assay. Control: wild type epimastigote; blue: hoechst staining of nucleus (n) and kinetoplast (k) DNA; red: PAT (arrow) and PPT labeling with AlexaFluor 594. Bars = 5 µm.
    Alexafluor 594, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 583 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexafluor 594/product/Thermo Fisher
    Average 90 stars, based on 583 article reviews
    Price from $9.99 to $1999.99
    alexafluor 594 - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    N/A
    The carboxylic acid of 5 ROX is used for oligonucleotide labeling and automated DNA sequencing applications Conjugates of this dye have longer wavelength spectra than the spectra of Lisaamine rhodamine
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    Image Search Results


    localisation of FLAG tagged PATs and PPTs in  Trypanosoma cruzi  epimastigotes by immunofluorescence assay. Control: wild type epimastigote; blue: hoechst staining of nucleus (n) and kinetoplast (k) DNA; red: PAT (arrow) and PPT labeling with AlexaFluor 594. Bars = 5 µm.

    Journal: Memórias do Instituto Oswaldo Cruz

    Article Title: Subcellular localisation of FLAG tagged enzymes of the dynamic protein S-palmitoylation cycle of Trypanosoma cruzi epimastigotes

    doi: 10.1590/0074-02760180086

    Figure Lengend Snippet: localisation of FLAG tagged PATs and PPTs in Trypanosoma cruzi epimastigotes by immunofluorescence assay. Control: wild type epimastigote; blue: hoechst staining of nucleus (n) and kinetoplast (k) DNA; red: PAT (arrow) and PPT labeling with AlexaFluor 594. Bars = 5 µm.

    Article Snippet: After three washes in PBS, the samples were incubated in the same conditions with a secondary goat anti-mouse antibody coupled to AlexaFluor 594 (Thermo Fischer Scientific, Waltham, MA, USA) diluted 1:600 in incubation buffer.

    Techniques: Immunofluorescence, Staining, Labeling

    Endocytosis and Phagocytosis of Bacteria by IPSDMs and PBDMs (A) Representative images of the AcLDL-Alexa Fluor 594 uptake assay by different subtypes of IPSDMs and PBDMs. AcLDL positive uptake is shown in red; cell nuclei are stained with Hoechst in blue. Scale bar represents 100 μm. (B) Quantification of AcLDL-Alexa Fluor 594 median fluorescence intensity of different macrophage subtypes by FACS. Error bars are ±SD of three independent experiments. Uncorrected Fisher's least significant differences test: ns, non-significant; ∗ p

    Journal: Stem Cell Reports

    Article Title: Differentiation and Functional Comparison of Monocytes and Macrophages from hiPSCs with Peripheral Blood Derivatives

    doi: 10.1016/j.stemcr.2019.05.003

    Figure Lengend Snippet: Endocytosis and Phagocytosis of Bacteria by IPSDMs and PBDMs (A) Representative images of the AcLDL-Alexa Fluor 594 uptake assay by different subtypes of IPSDMs and PBDMs. AcLDL positive uptake is shown in red; cell nuclei are stained with Hoechst in blue. Scale bar represents 100 μm. (B) Quantification of AcLDL-Alexa Fluor 594 median fluorescence intensity of different macrophage subtypes by FACS. Error bars are ±SD of three independent experiments. Uncorrected Fisher's least significant differences test: ns, non-significant; ∗ p

    Article Snippet: Alexa Fluor 594 AcLDL (Thermo Fisher Scientific) was used for the AcLDL uptake assay following the manufacturer's instructions.

    Techniques: Staining, Fluorescence, FACS

    In vitro analysis of human B12L and rat mAb35. a) Competitive binding of rat mAb35 was monitored by flow cytometer-based binding assay. The indicated concentrations of Abs (mAb35xich1, B12L and Ctrl IgG) were added to DB40 cells prior to spiking with 10 μg/mL of rat mAb35. Data was obtained by monitoring the mean fluorescence intensity (MFI) of anti-rat IgG-PE signal. The percent inhibition was calculated with the following formula: (MFI of rat mAb35 signal without blocking Abs–MFI of rat mAb35 signal with blocking Abs) / (MFI of rat mAb35 signal without blocking Abs–background signal) x 100 (%). All data were obtained from experiments performed in triplicate and are presented as mean values with SD. Ctrl IgG: isotype-matched human IgG for negative ctrl IgG. b) Agonistic and c) antagonistic activities of B12L/mAb35 were monitored by Ca ++ influx in DB40 cells. Acetylcholine (ACh) was used as a positive control agonist. Atropine was used as an inhibitor of muscarinic type AChR to reduce the background signal for antagonist activity. α-Btx was used as a positive control antagonist. d) Downmodulation of nAChRs induced by B12L, mAb35xich1, and their Fabs was monitored by fluorescence signal of the α-Btx-Alexa Fluor 488 probe. Binding at 100% represents the maximum fluorescence signal of α-Btx-Alexa Fluor 488 bound to nAChRs on the surface of DB40 cells. BG: background signal. Statistical analysis among groups in a) and d) were conducted using Student’s t-test, with p

    Journal: PLoS ONE

    Article Title: Analysis of peripheral B cells and autoantibodies against the anti-nicotinic acetylcholine receptor derived from patients with myasthenia gravis using single-cell manipulation tools

    doi: 10.1371/journal.pone.0185976

    Figure Lengend Snippet: In vitro analysis of human B12L and rat mAb35. a) Competitive binding of rat mAb35 was monitored by flow cytometer-based binding assay. The indicated concentrations of Abs (mAb35xich1, B12L and Ctrl IgG) were added to DB40 cells prior to spiking with 10 μg/mL of rat mAb35. Data was obtained by monitoring the mean fluorescence intensity (MFI) of anti-rat IgG-PE signal. The percent inhibition was calculated with the following formula: (MFI of rat mAb35 signal without blocking Abs–MFI of rat mAb35 signal with blocking Abs) / (MFI of rat mAb35 signal without blocking Abs–background signal) x 100 (%). All data were obtained from experiments performed in triplicate and are presented as mean values with SD. Ctrl IgG: isotype-matched human IgG for negative ctrl IgG. b) Agonistic and c) antagonistic activities of B12L/mAb35 were monitored by Ca ++ influx in DB40 cells. Acetylcholine (ACh) was used as a positive control agonist. Atropine was used as an inhibitor of muscarinic type AChR to reduce the background signal for antagonist activity. α-Btx was used as a positive control antagonist. d) Downmodulation of nAChRs induced by B12L, mAb35xich1, and their Fabs was monitored by fluorescence signal of the α-Btx-Alexa Fluor 488 probe. Binding at 100% represents the maximum fluorescence signal of α-Btx-Alexa Fluor 488 bound to nAChRs on the surface of DB40 cells. BG: background signal. Statistical analysis among groups in a) and d) were conducted using Student’s t-test, with p

    Article Snippet: Small pieces of these fibers were washed with 0.1M glycine/PBS for 30 min and blocked with 2% BSA/PBS for 60 min. To analyze complement deposition, muscle fibers were incubated with Alexa Fluor 594-α-Btx (Thermo Fisher Scientific) diluted 1:1000 in 2% BSA/PBS and mouse Ab against rat C3 (clone 12E2, Novus Biologicals, 1 μg/mL final concentration) for 16 h at 4°C.

    Techniques: In Vitro, Binding Assay, Flow Cytometry, Cytometry, Fluorescence, Inhibition, Blocking Assay, Positive Control, Activity Assay

    Transient heme-iron conditioning of NTHI alters trafficking to early endosomes. (A) NHBE cells were cocultured with either transiently restricted (TR) or continuously exposed (CE) NTHI strain 86-028NP(pGM1.1) for 4 h. Epithelial cell membranes were labeled with wheat germ agglutinin conjugated to Alexa Fluor 350 (blue), and bacteria were visualized by GFP fluorescence (green). Early endosomes were labeled with rabbit antibody to early endosomal antigen 1 (EEA1) protein and visualized with donkey anti-rabbit IgG conjugated to Alexa Fluor 594 (red). Representative images are shown for each condition (TR or CE) with individual and merged fluorescence images shown for depiction of colocalization. Colocalization of bacteria with EEA1 is observed as either yellow (merged) or red EEA1 label closely surrounding clusters of green NTHI bacteria. Bar, 10 µm. (B) Additional images representative of those depicted in panel A. (C) The number of colocalization events per cell was quantified by visual assessment of 200 individually infected cells. Statistical significance was determined by Mann-Whitney U test, and error bars represent standard errors of the means. (D and E) Transmission electron microscopy of NHBE cells cocultured with TR or CE conditioned NTHI for 4 h and subsequently immunolabeled to detect bacterial association with EEA1. Early endosomes were labeled with rabbit antibody to EEA1 and visualized with anti-rabbit IgG antibody conjugated to an 18-nm colloidal gold particle. EEA1-containing vesicles devoid of bacteria (labeled B) are indicated by yellow arrows, while red arrows indicate EEA1-containing vesicles associated with bacteria. Bar, 500 nm.

    Journal: mSphere

    Article Title: Transient Nutrient Deprivation Promotes Macropinocytosis-Dependent Intracellular Bacterial Community Development

    doi: 10.1128/mSphere.00286-18

    Figure Lengend Snippet: Transient heme-iron conditioning of NTHI alters trafficking to early endosomes. (A) NHBE cells were cocultured with either transiently restricted (TR) or continuously exposed (CE) NTHI strain 86-028NP(pGM1.1) for 4 h. Epithelial cell membranes were labeled with wheat germ agglutinin conjugated to Alexa Fluor 350 (blue), and bacteria were visualized by GFP fluorescence (green). Early endosomes were labeled with rabbit antibody to early endosomal antigen 1 (EEA1) protein and visualized with donkey anti-rabbit IgG conjugated to Alexa Fluor 594 (red). Representative images are shown for each condition (TR or CE) with individual and merged fluorescence images shown for depiction of colocalization. Colocalization of bacteria with EEA1 is observed as either yellow (merged) or red EEA1 label closely surrounding clusters of green NTHI bacteria. Bar, 10 µm. (B) Additional images representative of those depicted in panel A. (C) The number of colocalization events per cell was quantified by visual assessment of 200 individually infected cells. Statistical significance was determined by Mann-Whitney U test, and error bars represent standard errors of the means. (D and E) Transmission electron microscopy of NHBE cells cocultured with TR or CE conditioned NTHI for 4 h and subsequently immunolabeled to detect bacterial association with EEA1. Early endosomes were labeled with rabbit antibody to EEA1 and visualized with anti-rabbit IgG antibody conjugated to an 18-nm colloidal gold particle. EEA1-containing vesicles devoid of bacteria (labeled B) are indicated by yellow arrows, while red arrows indicate EEA1-containing vesicles associated with bacteria. Bar, 500 nm.

    Article Snippet: Host cell membranes were visualized using wheat germ agglutinin (WGA) conjugated with Alexa Fluor 594 (final concentration, 5 µg/ml; Thermo Fisher Scientific), and DNA was visualized with Hoechst 33342 (Thermo Fisher Scientific).

    Techniques: Labeling, Fluorescence, Infection, MANN-WHITNEY, Transmission Assay, Electron Microscopy, Immunolabeling

    Transiently restricted NTHI invades and survives within human epithelial cells in intracellular bacterial communities. (A) Normal human bronchial epithelial (NHBE) cells were cocultured with either transiently restricted (TR) or continuously exposed (CE) NTHI for 1 h, and total bacterial association (no gentamicin) or intracellular bacteria only (+ gentamicin) were quantified. Results are depicted as the percentage of inoculum that remained viable with or without gentamicin treatment. Statistical significance was determined using a two-tailed Student t test of the means from duplicate wells from each of three biological replicates. Error bars indicate standard errors of the means. ns, not significant. (B) The invasion index of transiently restricted (TR) and continuously exposed (CE) NTHI defined as the number of viable intracellular bacteria when normalized for total association. Statistical significance was determined using a two-tailed Student t test on the mean from technical duplicates from each of three biological replicates. Error bars represent standard errors of the means. (C) Intracellular survival of transiently restricted (TR) and continuously exposed (CE) NTHI following infection of NHBE cells for 2 or 24 h. Results are depicted as the percentage of inoculum that remained viable following gentamicin treatment. Statistical significance was determined using a two-tailed Student t test on the mean from duplicates from each of eight biological replicates for the 2-h time point and five biological replicates for the 24-h time point. Error bars represent standard errors of the means. (D to G) NTHI strain 86-028NP(pGM1.1) expressing green fluorescent protein was transiently restricted (TR) or continuously exposed (CE) to heme-iron and then cocultured with NHBE cells for 4 (D and E) or 24 (F and G) hours. Epithelial cell membranes were labeled with wheat germ agglutinin conjugated to Alexa Fluor 594 (red), and bacteria were visualized by GFP fluorescence (green). Bar, 10 µm. Images depict the formation of early IBCs by TR NTHI as early as 4 h postinoculation (D) that progress to mature IBCs at 24 h (F). CE NTHI localizes in circular compartments (E, inset) and does not form IBCs 24 h postinoculation (G).

    Journal: mSphere

    Article Title: Transient Nutrient Deprivation Promotes Macropinocytosis-Dependent Intracellular Bacterial Community Development

    doi: 10.1128/mSphere.00286-18

    Figure Lengend Snippet: Transiently restricted NTHI invades and survives within human epithelial cells in intracellular bacterial communities. (A) Normal human bronchial epithelial (NHBE) cells were cocultured with either transiently restricted (TR) or continuously exposed (CE) NTHI for 1 h, and total bacterial association (no gentamicin) or intracellular bacteria only (+ gentamicin) were quantified. Results are depicted as the percentage of inoculum that remained viable with or without gentamicin treatment. Statistical significance was determined using a two-tailed Student t test of the means from duplicate wells from each of three biological replicates. Error bars indicate standard errors of the means. ns, not significant. (B) The invasion index of transiently restricted (TR) and continuously exposed (CE) NTHI defined as the number of viable intracellular bacteria when normalized for total association. Statistical significance was determined using a two-tailed Student t test on the mean from technical duplicates from each of three biological replicates. Error bars represent standard errors of the means. (C) Intracellular survival of transiently restricted (TR) and continuously exposed (CE) NTHI following infection of NHBE cells for 2 or 24 h. Results are depicted as the percentage of inoculum that remained viable following gentamicin treatment. Statistical significance was determined using a two-tailed Student t test on the mean from duplicates from each of eight biological replicates for the 2-h time point and five biological replicates for the 24-h time point. Error bars represent standard errors of the means. (D to G) NTHI strain 86-028NP(pGM1.1) expressing green fluorescent protein was transiently restricted (TR) or continuously exposed (CE) to heme-iron and then cocultured with NHBE cells for 4 (D and E) or 24 (F and G) hours. Epithelial cell membranes were labeled with wheat germ agglutinin conjugated to Alexa Fluor 594 (red), and bacteria were visualized by GFP fluorescence (green). Bar, 10 µm. Images depict the formation of early IBCs by TR NTHI as early as 4 h postinoculation (D) that progress to mature IBCs at 24 h (F). CE NTHI localizes in circular compartments (E, inset) and does not form IBCs 24 h postinoculation (G).

    Article Snippet: Host cell membranes were visualized using wheat germ agglutinin (WGA) conjugated with Alexa Fluor 594 (final concentration, 5 µg/ml; Thermo Fisher Scientific), and DNA was visualized with Hoechst 33342 (Thermo Fisher Scientific).

    Techniques: Two Tailed Test, Infection, Expressing, Labeling, Fluorescence

    Pharmacological inhibition of endocytosis pathways reveals that nutritionally conditioned NTHI is internalized into cells through multiple mechanisms. (A) Representative images depicting inhibition of uptake of known fluorescent cargo conjugates (Alexa Fluor 488, green) into NHBE cells in the presence of each pharmacological inhibitor [CPZ, chlorpromazine; MβCD, methyl-β-cyclodextrin; EIPA, 5-( N -ethyl- N -isopropyl)-amiloride]. The optimal concentration of cytochalasin D (CytoD) was determined by staining F-actin with phalloidin conjugated to Alexa Fluor 350. Epithelial cell membranes were labeled with wheat germ agglutinin conjugated to Alexa Fluor 594 (red). Bar, 10 µm. (B) Fluorescence microscopy was used to determine uptake of transiently restricted (TR) or continuously exposed (CE) NTHI strain 86-028NP(pGM1.1) following pharmacological inhibition of endocytosis pathways. NHBE cells were pretreated with pharmacological inhibitors prior to a 4-h incubation with TR or CE NTHI strain 86-028NP(pGM1.1). NTHI was visualized by GFP fluorescence (green), and epithelial cell membranes were labeled with wheat germ agglutinin conjugated to Alexa Fluor 594 (red). The far-right panel depicts infected cells with no inhibitor (Untreated) for comparison. Each experiment was performed in three biological replicates, and representative images are shown. Bar, 10 µm. (C) Viable intracellular bacteria were enumerated following gentamicin treatment of NHBE cells that were incubated with transiently restricted (TR) or continuously exposed (CE) NTHI for 1 h in the presence of pharmacological inhibitors compared to untreated controls. Statistical significance was determined by analysis of variance with means from duplicate wells from three independent biological replicates, and error bars represent standard errors of the means (**, P

    Journal: mSphere

    Article Title: Transient Nutrient Deprivation Promotes Macropinocytosis-Dependent Intracellular Bacterial Community Development

    doi: 10.1128/mSphere.00286-18

    Figure Lengend Snippet: Pharmacological inhibition of endocytosis pathways reveals that nutritionally conditioned NTHI is internalized into cells through multiple mechanisms. (A) Representative images depicting inhibition of uptake of known fluorescent cargo conjugates (Alexa Fluor 488, green) into NHBE cells in the presence of each pharmacological inhibitor [CPZ, chlorpromazine; MβCD, methyl-β-cyclodextrin; EIPA, 5-( N -ethyl- N -isopropyl)-amiloride]. The optimal concentration of cytochalasin D (CytoD) was determined by staining F-actin with phalloidin conjugated to Alexa Fluor 350. Epithelial cell membranes were labeled with wheat germ agglutinin conjugated to Alexa Fluor 594 (red). Bar, 10 µm. (B) Fluorescence microscopy was used to determine uptake of transiently restricted (TR) or continuously exposed (CE) NTHI strain 86-028NP(pGM1.1) following pharmacological inhibition of endocytosis pathways. NHBE cells were pretreated with pharmacological inhibitors prior to a 4-h incubation with TR or CE NTHI strain 86-028NP(pGM1.1). NTHI was visualized by GFP fluorescence (green), and epithelial cell membranes were labeled with wheat germ agglutinin conjugated to Alexa Fluor 594 (red). The far-right panel depicts infected cells with no inhibitor (Untreated) for comparison. Each experiment was performed in three biological replicates, and representative images are shown. Bar, 10 µm. (C) Viable intracellular bacteria were enumerated following gentamicin treatment of NHBE cells that were incubated with transiently restricted (TR) or continuously exposed (CE) NTHI for 1 h in the presence of pharmacological inhibitors compared to untreated controls. Statistical significance was determined by analysis of variance with means from duplicate wells from three independent biological replicates, and error bars represent standard errors of the means (**, P

    Article Snippet: Host cell membranes were visualized using wheat germ agglutinin (WGA) conjugated with Alexa Fluor 594 (final concentration, 5 µg/ml; Thermo Fisher Scientific), and DNA was visualized with Hoechst 33342 (Thermo Fisher Scientific).

    Techniques: Inhibition, Concentration Assay, Staining, Labeling, Fluorescence, Microscopy, Incubation, Infection

    Inhibition of macropinocytosis redirects transiently restricted NTHI to the endolysosomal pathway and decreases intracellular survival. (A to D) NHBE cells were pretreated with 60 µM EIPA prior to coculture with transiently restricted (TR) or continuously exposed (CE) NTHI strain 86-028NP(pGM1.1) and visualized for colocalization of NTHI with EEA1 by fluorescence microscopy. NTHI was visualized by GFP fluorescence (green), EEA1 was labeled with rabbit antibody to EEA1 and visualized with donkey anti-rabbit IgG conjugated to Alexa Fluor 594 (red), and epithelial cell membranes were visualized with wheat germ agglutinin conjugated to Alexa Fluor 350 (blue). Representative images depict colocalization of TR (A) or CE (C) NTHI with EEA1 as observed by yellow fluorescence or red EEA1 labeling closely surrounding clusters of green NTHI bacteria. Bar, 10 µm. (B) EIPA pretreatment of NHBE cells significantly increases the number of colocalization events of TR NTHI with EEA1 in infected cells compared to infected cells that were not treated with EIPA. (D) Colocalization of CE NTHI with EEA1 does not significantly change in the presence or absence of EIPA. Statistical significance was determined by Mann-Whitney U test of colocalization events from a total of 200 independent cells from three biological assays. (E) Viable intracellular bacteria were enumerated following gentamicin treatment of NHBE cells infected with TR or CE NTHI in the presence and absence of EIPA. Statistical significance was determined by two-tailed Student’s t test of the means for duplicate wells from three independent biological replicates, and error bars represent standard errors of the means. (F to H) Intracellular bacterial communities were enumerated in chinchilla middle ear epithelial cells incubated with TR NTHI in the presence or absence of EIPA. (F and G) Bacteria were visualized by GFP fluorescence (green), epithelial cell members were stained with wheat germ agglutinin conjugated to Alex Fluor 594 (red), and host and bacterial DNA were labeled with Hoechst stain (blue). Bar, 10 µm. (H) Statistical significance was determined by two-tailed Student’s t test of the mean from duplicate wells from each of three independent biological replicates, and error bars represent standard errors of the means.

    Journal: mSphere

    Article Title: Transient Nutrient Deprivation Promotes Macropinocytosis-Dependent Intracellular Bacterial Community Development

    doi: 10.1128/mSphere.00286-18

    Figure Lengend Snippet: Inhibition of macropinocytosis redirects transiently restricted NTHI to the endolysosomal pathway and decreases intracellular survival. (A to D) NHBE cells were pretreated with 60 µM EIPA prior to coculture with transiently restricted (TR) or continuously exposed (CE) NTHI strain 86-028NP(pGM1.1) and visualized for colocalization of NTHI with EEA1 by fluorescence microscopy. NTHI was visualized by GFP fluorescence (green), EEA1 was labeled with rabbit antibody to EEA1 and visualized with donkey anti-rabbit IgG conjugated to Alexa Fluor 594 (red), and epithelial cell membranes were visualized with wheat germ agglutinin conjugated to Alexa Fluor 350 (blue). Representative images depict colocalization of TR (A) or CE (C) NTHI with EEA1 as observed by yellow fluorescence or red EEA1 labeling closely surrounding clusters of green NTHI bacteria. Bar, 10 µm. (B) EIPA pretreatment of NHBE cells significantly increases the number of colocalization events of TR NTHI with EEA1 in infected cells compared to infected cells that were not treated with EIPA. (D) Colocalization of CE NTHI with EEA1 does not significantly change in the presence or absence of EIPA. Statistical significance was determined by Mann-Whitney U test of colocalization events from a total of 200 independent cells from three biological assays. (E) Viable intracellular bacteria were enumerated following gentamicin treatment of NHBE cells infected with TR or CE NTHI in the presence and absence of EIPA. Statistical significance was determined by two-tailed Student’s t test of the means for duplicate wells from three independent biological replicates, and error bars represent standard errors of the means. (F to H) Intracellular bacterial communities were enumerated in chinchilla middle ear epithelial cells incubated with TR NTHI in the presence or absence of EIPA. (F and G) Bacteria were visualized by GFP fluorescence (green), epithelial cell members were stained with wheat germ agglutinin conjugated to Alex Fluor 594 (red), and host and bacterial DNA were labeled with Hoechst stain (blue). Bar, 10 µm. (H) Statistical significance was determined by two-tailed Student’s t test of the mean from duplicate wells from each of three independent biological replicates, and error bars represent standard errors of the means.

    Article Snippet: Host cell membranes were visualized using wheat germ agglutinin (WGA) conjugated with Alexa Fluor 594 (final concentration, 5 µg/ml; Thermo Fisher Scientific), and DNA was visualized with Hoechst 33342 (Thermo Fisher Scientific).

    Techniques: Inhibition, Fluorescence, Microscopy, Labeling, Infection, MANN-WHITNEY, Two Tailed Test, Incubation, Staining