triethylammonium  (Thermo Fisher)


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    Name:
    NBD PE N 7 Nitrobenz 2 Oxa 1 3 Diazol 4 yl 1 2 Dihexadecanoyl sn Glycero 3 Phosphoethanolamine Triethylammonium Salt
    Description:
    The phospholipid NBD PE is labeled on the head group with the environment sensitive fluorophore NBD excitation emission maxima 463 536 nm
    Catalog Number:
    n360
    Price:
    None
    Applications:
    Cell Analysis|Cellular Imaging|Immunofluorescence (IF)|Immunofluorescence Staining & Detection
    Category:
    Labeling Detection Products
    Buy from Supplier


    Structured Review

    Thermo Fisher triethylammonium
    The phospholipid NBD PE is labeled on the head group with the environment sensitive fluorophore NBD excitation emission maxima 463 536 nm
    https://www.bioz.com/result/triethylammonium/product/Thermo Fisher
    Average 77 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    triethylammonium - by Bioz Stars, 2020-02
    77/100 stars

    Images

    Related Articles

    High Performance Liquid Chromatography:

    Article Title: Physical effects underlying the transition from primitive to modern cell membranes
    Article Snippet: Rhodamine DHPE (Rhodamine B 1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine) and NBD-PE [ N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl- sn - glycero-3-phosphoethanolamine] were obtained from Invitrogen. .. The imidazolide was purified by reverse-phase HPLC on a C18 column (Alltech) equilibrated with 20 mM triethylammonium bicarbonate pH 7.8, 2% acetonitrile and eluted with a 2–9% gradient of acetonitrile.

    Electron Microscopy:

    Article Title: Visualization of the Two-Step Fusion Process of the Retrovirus Avian Sarcoma/Leukosis Virus by Cryo-Electron Tomography
    Article Snippet: To produce fluorescent liposomes, lipid mixtures of phosphatidylcholine (PC; number catalog 830051P; Avanti), phosphatidylethanolamine (PE; catalog number 841118P; Avanti), sphingomyelin (SPH; 860061P; Avanti), cholesterol (CH; C-3137; Sigma), rhodamine phosphatidylethanolamine (RH-PE; 810146; Avanti), and ( N -7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl- sn -glycero-3-phosphatidylethanolamine (NBD-PE; N-360; Molecular Probes) at a ratio of 1:1:1:1.5:0.045:0.11 were dried with N2 , lyophilized overnight, and resuspended in HM buffer to a 2 mM lipid concentration. .. Liposomes for electron microscopy were produced as described above, except that PC, PE, SPH, and CH were mixed in a 1:1:1:1.5 ratio and resuspended at a 20 mM lipid concentration.

    Transfection:

    Article Title: The binding capability of plasma phospholipid transfer protein, but not HDL pool size, is critical to repress LPS induced inflammation
    Article Snippet: Fetal bovine serum (FBS), 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), and lipofectamine 2000 transfection kit were purchased from Life technology (Grand Island, NY, USA). .. NBD-labelled phosphoethanolamine (NBD-PE, N-360) was purchased from Molecular Probe (Life technologies, USA).

    Recombinant:

    Article Title: The binding capability of plasma phospholipid transfer protein, but not HDL pool size, is critical to repress LPS induced inflammation
    Article Snippet: LPS (Cat. No. L2880), thioglycollate broth (Cat. No. 70157), recombinant tumor necrosis factor α (Cat. No. T0157), Glyburide (Cat. No. G2539), LPS from E. Coli O55:B5 (Cat. No. L2880), LPS-FITC from E. Coli O55:B5 (Cat. No. F8666), PLTP siRNA, and control siRNA were purchased from Sigma-Aldrich (St. Louis, MO, USA). .. NBD-labelled phosphoethanolamine (NBD-PE, N-360) was purchased from Molecular Probe (Life technologies, USA).

    Filtration:

    Article Title: Droplet Shape Analysis and Permeability Studies in Droplet Lipid Bilayers
    Article Snippet: The fluorescent lipid probe, NBD-PE (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) was purchased from Invitrogen Corporation. .. Deionized water was used for the preparation of all buffers after filtration using a 0.2-μm filter.

    Fluorescence:

    Article Title: Real-time Visualization of Dynamin-catalyzed Membrane Fission and Vesicle Release
    Article Snippet: .. Fluorescence intensity of liposomes and SUPER templates containing 2 mol% NBD-PE (Invitrogen) and SUPER templates made from such liposomes was recorded before and after adding 10 μl of 1 M dithionite (Sigma) stock solution prepared in 1 M Tris pH 10.0 buffer, while being continuously stirred. .. Fluorescence intensities were acquired in a Fluorolog-3 steady-state spectrofluorometer (Horiba Jobin Yvon) at excitation and emission wavelengths of 470 nm (2 nm bandpass) and 540 nm (4 nm bandpass), respectively, in a temperature-controlled 1 cm × 1 cm quartz cuvette at 25 °C.

    Article Title: Interactions of apomyoglobin with membranes: Mechanisms and effects on heme uptake
    Article Snippet: Trp fluorescence was used as the probe of protein concentration. .. Vesicles were prepared with 0.5% of NBD-phosphatidyl ethanolamine (NBD-PE) (N-360, Molecular Probes).

    MTT Assay:

    Article Title: The binding capability of plasma phospholipid transfer protein, but not HDL pool size, is critical to repress LPS induced inflammation
    Article Snippet: Fetal bovine serum (FBS), 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), and lipofectamine 2000 transfection kit were purchased from Life technology (Grand Island, NY, USA). .. NBD-labelled phosphoethanolamine (NBD-PE, N-360) was purchased from Molecular Probe (Life technologies, USA).

    Protease Inhibitor:

    Article Title: Prodomain of Furin Promotes Phospholipid Transfer Protein Proteasomal Degradation in Hepatocytes
    Article Snippet: Complete protease inhibitor cocktail tablets were purchased from Roche (05892970001). .. Nitrobenzoxadiazole (NBD)‐labeled phosphoethanolamine (N‐360) was purchased from Molecular Probe (Life Technologies).

    Article Title: The binding capability of plasma phospholipid transfer protein, but not HDL pool size, is critical to repress LPS induced inflammation
    Article Snippet: Complete protease inhibitor cocktail tablets (Roche 05892970001) and PhoStop cocktail (Roche 04906845001) were purchased from Roche (Schweiz, Germany). .. NBD-labelled phosphoethanolamine (NBD-PE, N-360) was purchased from Molecular Probe (Life technologies, USA).

    Centrifugation:

    Article Title: Interactions of apomyoglobin with membranes: Mechanisms and effects on heme uptake
    Article Snippet: Paragraph title: Partitioning of aMb to LUVs monitored by centrifugation ... Vesicles were prepared with 0.5% of NBD-phosphatidyl ethanolamine (NBD-PE) (N-360, Molecular Probes).

    Purification:

    Article Title: Physical effects underlying the transition from primitive to modern cell membranes
    Article Snippet: Rhodamine DHPE (Rhodamine B 1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine) and NBD-PE [ N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl- sn - glycero-3-phosphoethanolamine] were obtained from Invitrogen. .. The imidazolide was purified by reverse-phase HPLC on a C18 column (Alltech) equilibrated with 20 mM triethylammonium bicarbonate pH 7.8, 2% acetonitrile and eluted with a 2–9% gradient of acetonitrile.

    Produced:

    Article Title: Visualization of the Two-Step Fusion Process of the Retrovirus Avian Sarcoma/Leukosis Virus by Cryo-Electron Tomography
    Article Snippet: To produce fluorescent liposomes, lipid mixtures of phosphatidylcholine (PC; number catalog 830051P; Avanti), phosphatidylethanolamine (PE; catalog number 841118P; Avanti), sphingomyelin (SPH; 860061P; Avanti), cholesterol (CH; C-3137; Sigma), rhodamine phosphatidylethanolamine (RH-PE; 810146; Avanti), and ( N -7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl- sn -glycero-3-phosphatidylethanolamine (NBD-PE; N-360; Molecular Probes) at a ratio of 1:1:1:1.5:0.045:0.11 were dried with N2 , lyophilized overnight, and resuspended in HM buffer to a 2 mM lipid concentration. .. Liposomes for electron microscopy were produced as described above, except that PC, PE, SPH, and CH were mixed in a 1:1:1:1.5 ratio and resuspended at a 20 mM lipid concentration.

    Activation Assay:

    Article Title: Physical effects underlying the transition from primitive to modern cell membranes
    Article Snippet: Rhodamine DHPE (Rhodamine B 1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine) and NBD-PE [ N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl- sn - glycero-3-phosphoethanolamine] were obtained from Invitrogen. .. 3 H-ImpdA was prepared by activation of 2,8-3 H dAMP (Moravek Biochemicals) with carbonyldiimidazole (CDI) ( ).

    Incubation:

    Article Title: Interactions of apomyoglobin with membranes: Mechanisms and effects on heme uptake
    Article Snippet: Vesicles were prepared with 0.5% of NBD-phosphatidyl ethanolamine (NBD-PE) (N-360, Molecular Probes). .. LUVs and proteins were incubated in 5 mL for 2 h at room temperature.

    other:

    Article Title: Concentration-Driven Growth of Model Protocell Membranes
    Article Snippet: Laurdan, NBD-PE (N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine), and Rhodamine-DHPE (Rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine) were obtained from Invitrogen.

    Article Title: WDFY2 restrains matrix metalloproteinase secretion and cell invasion by controlling VAMP3-dependent recycling
    Article Snippet: Liposome flotation assays Liposome flotation assays were designed similar to published procedures and performed as follows: Lipids (47% PC, 25% PE, 9% cholesterol, 10% PS, 5% PI (Avanti Polar Lipids), 5% PtdIns3P (Echelon), and 0.2% NBD-PE (Thermo Fisher), all % are molar %) were dissolved in Chloroform.

    Article Title: Characterization of a Membrane-active Peptide from the Bordetella pertussis CyaA Toxin *
    Article Snippet: ANTS (A-350), DPX (X-1525), and N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine, triethylammonium salt (N-360) were purchased from Molecular Probes (Eugene, OR).

    Article Title: Type 3 Secretion Translocators Spontaneously Assemble a Hexadecameric Transmembrane Complex *
    Article Snippet: NBD-PE ( N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine) was purchased from Life Technologies.

    Article Title: Identification of a Region That Assists Membrane Insertion and Translocation of the Catalytic Domain of Bordetella pertussis CyaA Toxin
    Article Snippet: 8-Anilino-1-naphthalene sulfonic acid (ANS) (A-47), ANTS (A-350), DPX (X-1525), and N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl- sn -glycero-3-phosphoethanolamine (N-360) were purchased from Molecular Probes (Eugene, OR).

    Spectroscopy:

    Article Title: Real-time Visualization of Dynamin-catalyzed Membrane Fission and Vesicle Release
    Article Snippet: Paragraph title: Fluorescence Spectroscopy and Dithionite Quenching Experiments ... Fluorescence intensity of liposomes and SUPER templates containing 2 mol% NBD-PE (Invitrogen) and SUPER templates made from such liposomes was recorded before and after adding 10 μl of 1 M dithionite (Sigma) stock solution prepared in 1 M Tris pH 10.0 buffer, while being continuously stirred.

    Protein Concentration:

    Article Title: Interactions of apomyoglobin with membranes: Mechanisms and effects on heme uptake
    Article Snippet: Trp fluorescence was used as the probe of protein concentration. .. Vesicles were prepared with 0.5% of NBD-phosphatidyl ethanolamine (NBD-PE) (N-360, Molecular Probes).

    Concentration Assay:

    Article Title: Visualization of the Two-Step Fusion Process of the Retrovirus Avian Sarcoma/Leukosis Virus by Cryo-Electron Tomography
    Article Snippet: .. To produce fluorescent liposomes, lipid mixtures of phosphatidylcholine (PC; number catalog 830051P; Avanti), phosphatidylethanolamine (PE; catalog number 841118P; Avanti), sphingomyelin (SPH; 860061P; Avanti), cholesterol (CH; C-3137; Sigma), rhodamine phosphatidylethanolamine (RH-PE; 810146; Avanti), and ( N -7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl- sn -glycero-3-phosphatidylethanolamine (NBD-PE; N-360; Molecular Probes) at a ratio of 1:1:1:1.5:0.045:0.11 were dried with N2 , lyophilized overnight, and resuspended in HM buffer to a 2 mM lipid concentration. ..

    Article Title: Interactions of apomyoglobin with membranes: Mechanisms and effects on heme uptake
    Article Snippet: The ratio L/P was equal to 300 and the aMb concentration 1 μM. .. Vesicles were prepared with 0.5% of NBD-phosphatidyl ethanolamine (NBD-PE) (N-360, Molecular Probes).

    Injection:

    Article Title: Nanoliposomes for encapsulation and delivery of the potential antitumoral methyl 6-methoxy-3-(4-methoxyphenyl)-1H-indole-2-carboxylate
    Article Snippet: Fluorescent-labelled lipids N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn -glycero-3-phosphoethanolamine (triethylammonium salt) (NBD-PE), 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn -glycero-3-phosphocholine (NBD-C6 -HPC), and 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn -glycero-3-phosphocholine (NBD-C12 -HPC) were obtained from Invitrogen (Carlsbad, CA, USA). .. Nanoliposomes were prepared by injection of an ethanolic solution of lipids/compound 1 mixture in an aqueous buffer solution under vigorous stirring, above the lipid melting transition temperature (ca .

    Binding Assay:

    Article Title: Real-time Visualization of Dynamin-catalyzed Membrane Fission and Vesicle Release
    Article Snippet: Fluorescence intensity of liposomes and SUPER templates containing 2 mol% NBD-PE (Invitrogen) and SUPER templates made from such liposomes was recorded before and after adding 10 μl of 1 M dithionite (Sigma) stock solution prepared in 1 M Tris pH 10.0 buffer, while being continuously stirred. .. All experiments were performed in 20 mM HEPES, pH 7.5, 150 mM NaCl buffer containing 10 μM unlabeled DOPC liposomes in order to prevent SUPER templates from binding and spilling their excess reservoir on to the walls of the cuvette.

    Generated:

    Article Title: Prodomain of Furin Promotes Phospholipid Transfer Protein Proteasomal Degradation in Hepatocytes
    Article Snippet: AdV‐profurin and AdV‐null were generated as previously described. .. Nitrobenzoxadiazole (NBD)‐labeled phosphoethanolamine (N‐360) was purchased from Molecular Probe (Life Technologies).

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  • 90
    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
    Thermo Scientific Triethylamonium bicarbonat TEAB is a 10X dissolution buffer for isobaric mass tag labeling experiments Related Products TMT10plex Isobaric Label Reagent Set 1 x 0 8 mg TMT10plex Isobaric
      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
      Buy from Supplier

    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