triethylammonium  (Thermo Fisher)


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  • 93
    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
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    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 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    triethylammonium - by Bioz Stars, 2020-09
    93/100 stars

    Images

    Related Articles

    Fluorescence:

    Article Title: Surface structure of the COPII-coated vesicle
    Article Snippet: .. The resulting solution was mixed with 2 μl of crosslinkers in a solution of dimethyl sulfoxide with or without NHS-biotin for 30 min on ice, and then the excess crosslinker was quenched by mixing 3 μl of 0.25 M Tris/1.9 M glycine, pH 8.2 and incubated at room temperature for 30 min. Proteins in this reaction were separated by SDS/PAGE and transferred to a polyvinylidene difluoride membrane, and the fluorescence of crosslinked NBD-PE was recorded by using a STORM860 image analyzer at a setting of 950 V. After recording, biotinylated proteins on the membrane were probed with a streptavidin-alkaline phosphatase conjugate (Pierce), and the activity of alkaline phosphatase was detected by using the Vistra ECF substrate (Amersham Pharmacia). .. The fluorescent product was detected by a STORM860 image analyzed at a setting of 650 V. Thereafter, proteins on the membrane were visualized by colloidal gold staining.

    CtB Assay:

    Article Title: Ganglioside embedded in reconstituted lipoprotein binds cholera toxin with elevated affinity [S]
    Article Snippet: .. 1,6-Diphenyl-1,3,5-hexatriene (DPH), Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethyl-ammonium salt (TR-DHPE), N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethyl-ammonium salt (NBD-PE), along with Alexa594-labeled cholera toxin subunit (CTB) were purchased from Invitrogen (Carlsbad, CA). .. Fluorescein-isothiocyanate (FITC)-labeled cholera toxin subunit B was obtained from Sigma Aldrich (Milwaukee, WI).

    Incubation:

    Article Title: MlaFEDB displays flippase activity to promote phospholipid transport towards the outer membrane of Gram-negative bacteria
    Article Snippet: .. 1 mg of MlaFEDB:NBD-PE was incubated with 0.05 mg/ml Proteinase K (Thermo Fisher) for 10 mins at 4 °C. .. In order to ensure the stability of the proteoliposomes, a control sample of MlaFEDB:NBD-PE was treated with 1 % (v/v) Triton X-100 before being subjected to Proteinase K digestion as outlined above.

    Article Title: Surface structure of the COPII-coated vesicle
    Article Snippet: .. The resulting solution was mixed with 2 μl of crosslinkers in a solution of dimethyl sulfoxide with or without NHS-biotin for 30 min on ice, and then the excess crosslinker was quenched by mixing 3 μl of 0.25 M Tris/1.9 M glycine, pH 8.2 and incubated at room temperature for 30 min. Proteins in this reaction were separated by SDS/PAGE and transferred to a polyvinylidene difluoride membrane, and the fluorescence of crosslinked NBD-PE was recorded by using a STORM860 image analyzer at a setting of 950 V. After recording, biotinylated proteins on the membrane were probed with a streptavidin-alkaline phosphatase conjugate (Pierce), and the activity of alkaline phosphatase was detected by using the Vistra ECF substrate (Amersham Pharmacia). .. The fluorescent product was detected by a STORM860 image analyzed at a setting of 650 V. Thereafter, proteins on the membrane were visualized by colloidal gold staining.

    other:

    Article Title: Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion [S]
    Article Snippet: N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn -glycero-3-phosphoethanolamine triethyl ammonium salt (NBD-PE) and N -((6-(biotinoyl)amino)hexanoyl-1,2-dihexadecanoyl-sn -glycerol-3-phosphoethanolaminetriethyl ammonium salt (Biotin-PE) were from Invitrogen (Carlsbad, CA).

    Activity Assay:

    Article Title: Surface structure of the COPII-coated vesicle
    Article Snippet: .. The resulting solution was mixed with 2 μl of crosslinkers in a solution of dimethyl sulfoxide with or without NHS-biotin for 30 min on ice, and then the excess crosslinker was quenched by mixing 3 μl of 0.25 M Tris/1.9 M glycine, pH 8.2 and incubated at room temperature for 30 min. Proteins in this reaction were separated by SDS/PAGE and transferred to a polyvinylidene difluoride membrane, and the fluorescence of crosslinked NBD-PE was recorded by using a STORM860 image analyzer at a setting of 950 V. After recording, biotinylated proteins on the membrane were probed with a streptavidin-alkaline phosphatase conjugate (Pierce), and the activity of alkaline phosphatase was detected by using the Vistra ECF substrate (Amersham Pharmacia). .. The fluorescent product was detected by a STORM860 image analyzed at a setting of 650 V. Thereafter, proteins on the membrane were visualized by colloidal gold staining.

    SDS Page:

    Article Title: Surface structure of the COPII-coated vesicle
    Article Snippet: .. The resulting solution was mixed with 2 μl of crosslinkers in a solution of dimethyl sulfoxide with or without NHS-biotin for 30 min on ice, and then the excess crosslinker was quenched by mixing 3 μl of 0.25 M Tris/1.9 M glycine, pH 8.2 and incubated at room temperature for 30 min. Proteins in this reaction were separated by SDS/PAGE and transferred to a polyvinylidene difluoride membrane, and the fluorescence of crosslinked NBD-PE was recorded by using a STORM860 image analyzer at a setting of 950 V. After recording, biotinylated proteins on the membrane were probed with a streptavidin-alkaline phosphatase conjugate (Pierce), and the activity of alkaline phosphatase was detected by using the Vistra ECF substrate (Amersham Pharmacia). .. The fluorescent product was detected by a STORM860 image analyzed at a setting of 650 V. Thereafter, proteins on the membrane were visualized by colloidal gold staining.

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  • 94
    Thermo Fisher biotin x dhpe lipids
    Single <t>Biotin-X-DHPE</t> molecules tagged with Streptavidin-Alexa546 were tracked on the curved supported lipid bilayers (ROC = 28 nm) in space and time. ( a ) example trajectories (blue) show the heterogeneous dynamics observed on both flat (white) and curved (black) regions. Scale bar = 1 µm; ( b ) the average step a molecule takes over 0.228 s (5 frames) when starting at a region of curvature (white) or at a flat region (grey); ( c ) the distribution of steps observed at curved and flat regions (shown in Figure S3 ) was fitted to Equation 3 to obtain the diffusion coefficients and the percentage of steps moving at that rate. The average D is plotted for t = 0.091, 0.228 and 0.456. Note that there is no fast component for the tracks that start at regions of curvature.
    Biotin X Dhpe Lipids, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotin x dhpe lipids/product/Thermo Fisher
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    99
    Thermo Fisher gdp bodipy fl
    OtDUB binds Rac1 and Cdc42 and catalyzes nucleotide exchange in vitro (a) Inputs and anti-Flag immunoprecipitates of lysates from HeLa cell ectopically expressing the indicated Flag-tagged OtDUB fragments. Proteins were resolved by SDS-PAGE and immunoblotted for Rho GTPases (Rac1,2,3; Cdc42; RhoA). (b) FLAG (top) and reciprocal GST (bottom) pulldown experiments between purified recombinant FLAG-tagged OtDUB fragments and GST-tagged Rac1 or Cdc42. Proteins were resolved by SDS-PAGE and stained with Coomassie Blue. (c) Time course of the dissociation of <t>BODIPY-GDP</t> from Rac1 or Cdc42 (9 μM) in the presence of OtDUB 275-1369 (0, 50, or 250 nM) as measured by loss of BODIPY-GDP fluorescence. Excitation and emission wavelength were 488 nm and 535 nm, respectively.
    Gdp Bodipy Fl, 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
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    Average 99 stars, based on 1 article reviews
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    99
    Thermo Fisher alexa fluor 594
    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 <t>Alexa</t> 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 <t>Fluor</t> 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.
    Alexa Fluor 594, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1873 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 1873 article reviews
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    Image Search Results


    Single Biotin-X-DHPE molecules tagged with Streptavidin-Alexa546 were tracked on the curved supported lipid bilayers (ROC = 28 nm) in space and time. ( a ) example trajectories (blue) show the heterogeneous dynamics observed on both flat (white) and curved (black) regions. Scale bar = 1 µm; ( b ) the average step a molecule takes over 0.228 s (5 frames) when starting at a region of curvature (white) or at a flat region (grey); ( c ) the distribution of steps observed at curved and flat regions (shown in Figure S3 ) was fitted to Equation 3 to obtain the diffusion coefficients and the percentage of steps moving at that rate. The average D is plotted for t = 0.091, 0.228 and 0.456. Note that there is no fast component for the tracks that start at regions of curvature.

    Journal: Membranes

    Article Title: Single Lipid Molecule Dynamics on Supported Lipid Bilayers with Membrane Curvature

    doi: 10.3390/membranes7010015

    Figure Lengend Snippet: Single Biotin-X-DHPE molecules tagged with Streptavidin-Alexa546 were tracked on the curved supported lipid bilayers (ROC = 28 nm) in space and time. ( a ) example trajectories (blue) show the heterogeneous dynamics observed on both flat (white) and curved (black) regions. Scale bar = 1 µm; ( b ) the average step a molecule takes over 0.228 s (5 frames) when starting at a region of curvature (white) or at a flat region (grey); ( c ) the distribution of steps observed at curved and flat regions (shown in Figure S3 ) was fitted to Equation 3 to obtain the diffusion coefficients and the percentage of steps moving at that rate. The average D is plotted for t = 0.091, 0.228 and 0.456. Note that there is no fast component for the tracks that start at regions of curvature.

    Article Snippet: The dynamics of Biotin-X-DHPE lipids were detected by in situ labeling with Alexa Fluor 546 (Thermo Fisher) conjugated to streptavidin (Strep-546).

    Techniques: Diffusion-based Assay

    OtDUB binds Rac1 and Cdc42 and catalyzes nucleotide exchange in vitro (a) Inputs and anti-Flag immunoprecipitates of lysates from HeLa cell ectopically expressing the indicated Flag-tagged OtDUB fragments. Proteins were resolved by SDS-PAGE and immunoblotted for Rho GTPases (Rac1,2,3; Cdc42; RhoA). (b) FLAG (top) and reciprocal GST (bottom) pulldown experiments between purified recombinant FLAG-tagged OtDUB fragments and GST-tagged Rac1 or Cdc42. Proteins were resolved by SDS-PAGE and stained with Coomassie Blue. (c) Time course of the dissociation of BODIPY-GDP from Rac1 or Cdc42 (9 μM) in the presence of OtDUB 275-1369 (0, 50, or 250 nM) as measured by loss of BODIPY-GDP fluorescence. Excitation and emission wavelength were 488 nm and 535 nm, respectively.

    Journal: bioRxiv

    Article Title: Crystal structure of a novel guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

    doi: 10.1101/2020.06.04.133876

    Figure Lengend Snippet: OtDUB binds Rac1 and Cdc42 and catalyzes nucleotide exchange in vitro (a) Inputs and anti-Flag immunoprecipitates of lysates from HeLa cell ectopically expressing the indicated Flag-tagged OtDUB fragments. Proteins were resolved by SDS-PAGE and immunoblotted for Rho GTPases (Rac1,2,3; Cdc42; RhoA). (b) FLAG (top) and reciprocal GST (bottom) pulldown experiments between purified recombinant FLAG-tagged OtDUB fragments and GST-tagged Rac1 or Cdc42. Proteins were resolved by SDS-PAGE and stained with Coomassie Blue. (c) Time course of the dissociation of BODIPY-GDP from Rac1 or Cdc42 (9 μM) in the presence of OtDUB 275-1369 (0, 50, or 250 nM) as measured by loss of BODIPY-GDP fluorescence. Excitation and emission wavelength were 488 nm and 535 nm, respectively.

    Article Snippet: BODIPY-GDP nucleotide exchange assays GTPases at a final concentration of 10 μM were loaded with sub-stoichiometric (1/4 [GTPase]) concentrations of GDP-BODIPY-FL (Thermo Scientific) in the presence of 50 mM Tris pH 7.5, 50 mM NaCl, 1 mM DTT, supplemented with 2 mM EDTA to promote unloading of co-purified nucleotide.

    Techniques: In Vitro, Expressing, SDS Page, Purification, Recombinant, Staining, Fluorescence

    A 200-residue OtDUB subdomain sufficient for binding Rac1 (a) OtDUB truncations used for mapping studies (top) and GST pulldown experiment using GST-OtDUB fragments as bait and Rac1 as prey (bottom). Gel was stained with Coomassie Blue. N-terminal truncation to residue 580 abolished binding, whereas the 548-759 fragment (OtDUB GEF domain) retained full binding capacity. (b) Size exclusion chromatography of OtDUB GEF :Rac1 mixtures demonstrates stable complex formation (top). Column fractions were evaluated by SDS-PAGE and protein staining (bottom). (c) Time course of the dissociation of BODIPY-GDP from Rac1 in the presence of increasing amounts of OtDUB GEF (residues 548-759). Raw fluorescence curves were fit to a single exponential decay (top), and initial rates were plotted against Rac1 concentration (bottom). Linear transformation of the titration yielded a k cat /K M of 2.6 ± 0.3 ×10 5 M -1 s -1 .

    Journal: bioRxiv

    Article Title: Crystal structure of a novel guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

    doi: 10.1101/2020.06.04.133876

    Figure Lengend Snippet: A 200-residue OtDUB subdomain sufficient for binding Rac1 (a) OtDUB truncations used for mapping studies (top) and GST pulldown experiment using GST-OtDUB fragments as bait and Rac1 as prey (bottom). Gel was stained with Coomassie Blue. N-terminal truncation to residue 580 abolished binding, whereas the 548-759 fragment (OtDUB GEF domain) retained full binding capacity. (b) Size exclusion chromatography of OtDUB GEF :Rac1 mixtures demonstrates stable complex formation (top). Column fractions were evaluated by SDS-PAGE and protein staining (bottom). (c) Time course of the dissociation of BODIPY-GDP from Rac1 in the presence of increasing amounts of OtDUB GEF (residues 548-759). Raw fluorescence curves were fit to a single exponential decay (top), and initial rates were plotted against Rac1 concentration (bottom). Linear transformation of the titration yielded a k cat /K M of 2.6 ± 0.3 ×10 5 M -1 s -1 .

    Article Snippet: BODIPY-GDP nucleotide exchange assays GTPases at a final concentration of 10 μM were loaded with sub-stoichiometric (1/4 [GTPase]) concentrations of GDP-BODIPY-FL (Thermo Scientific) in the presence of 50 mM Tris pH 7.5, 50 mM NaCl, 1 mM DTT, supplemented with 2 mM EDTA to promote unloading of co-purified nucleotide.

    Techniques: Binding Assay, Staining, Size-exclusion Chromatography, SDS Page, Fluorescence, Concentration Assay, Transformation Assay, Titration

    Biochemical function and structure of the OtDUB GEF :Rac1 complex (a) Three orthogonal views of the complex with OtDUB GEF in purple and Rac1 in cyan. Switch I and II loops are in salmon and yellow, respectively, and the β2-3 hairpin selectivity interface is green. (b) Close-up views of each key interface between OtDUB GEF and Rac1 showing selected residues, with hydrogen bonds and electrostatic interactions shown as dashes, water molecules as red spheres. (c) GST pulldown assays (left) of GST-OtDUB GEF (WT or charge-neutralizing mutations at each interface) incubated with Rac1 and analyzed by SDS-PAGE and Coomassie Blue staining. Corresponding BODIPY-GDP release assays are shown on the right. (d) Close-up views of each interface comparing the OtDUB GEF residues interacting with Rac1 (thick sticks) to the interactions made by other bacterial GEFs (thin sticks).

    Journal: bioRxiv

    Article Title: Crystal structure of a novel guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

    doi: 10.1101/2020.06.04.133876

    Figure Lengend Snippet: Biochemical function and structure of the OtDUB GEF :Rac1 complex (a) Three orthogonal views of the complex with OtDUB GEF in purple and Rac1 in cyan. Switch I and II loops are in salmon and yellow, respectively, and the β2-3 hairpin selectivity interface is green. (b) Close-up views of each key interface between OtDUB GEF and Rac1 showing selected residues, with hydrogen bonds and electrostatic interactions shown as dashes, water molecules as red spheres. (c) GST pulldown assays (left) of GST-OtDUB GEF (WT or charge-neutralizing mutations at each interface) incubated with Rac1 and analyzed by SDS-PAGE and Coomassie Blue staining. Corresponding BODIPY-GDP release assays are shown on the right. (d) Close-up views of each interface comparing the OtDUB GEF residues interacting with Rac1 (thick sticks) to the interactions made by other bacterial GEFs (thin sticks).

    Article Snippet: BODIPY-GDP nucleotide exchange assays GTPases at a final concentration of 10 μM were loaded with sub-stoichiometric (1/4 [GTPase]) concentrations of GDP-BODIPY-FL (Thermo Scientific) in the presence of 50 mM Tris pH 7.5, 50 mM NaCl, 1 mM DTT, supplemented with 2 mM EDTA to promote unloading of co-purified nucleotide.

    Techniques: Incubation, SDS Page, Staining

    OtDUB GEF utilizes a unique carbonyl “catalytic lever” to catalyze nucleotide exchange (a) Top: Structural overlay of GTPases from GDP-bound (PDB: 5N6O, grey) and GEF-bound structures: OtDUB GEF (cyan), SopE (red), Map (yellow), and TIAM1 (slate). GEFs are removed for clarity. Switch I residues Glu 62 , Ala 59 , and Lys 16 are shown as sticks. Arrows indicate the previously observed motions of the residues upon GEF binding. Bottom: BODIPY-GDP exchange assay reveals that Glu 62 is not required for efficient exchange catalyzed by OtDUB GEF . (b) Top: A loop of OtDUB GEF that interrupts α2 acts as a “catalytic lever” to promote release of GDP. OtDUB GEF is shown as cartoon (purple), and Rac1 is shown as transparent surface (cyan). GDP (not present in our structure) is modeled based on PDB 5N6O for reference. The steric clash and electrostatic repulsion are indicated by the red lines. Bottom: Nucleotide exchange assay reveals a strict requirement in the lever segment for exchange of BODIPY-GDP. (c) Calculated electrostatic surface potential map (unit k B T/e) shows high negative charge in the OtDUB GEF lever formed by a tri-carbonyl motif at the vicinity of the diphosphate group of GDP.

    Journal: bioRxiv

    Article Title: Crystal structure of a novel guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

    doi: 10.1101/2020.06.04.133876

    Figure Lengend Snippet: OtDUB GEF utilizes a unique carbonyl “catalytic lever” to catalyze nucleotide exchange (a) Top: Structural overlay of GTPases from GDP-bound (PDB: 5N6O, grey) and GEF-bound structures: OtDUB GEF (cyan), SopE (red), Map (yellow), and TIAM1 (slate). GEFs are removed for clarity. Switch I residues Glu 62 , Ala 59 , and Lys 16 are shown as sticks. Arrows indicate the previously observed motions of the residues upon GEF binding. Bottom: BODIPY-GDP exchange assay reveals that Glu 62 is not required for efficient exchange catalyzed by OtDUB GEF . (b) Top: A loop of OtDUB GEF that interrupts α2 acts as a “catalytic lever” to promote release of GDP. OtDUB GEF is shown as cartoon (purple), and Rac1 is shown as transparent surface (cyan). GDP (not present in our structure) is modeled based on PDB 5N6O for reference. The steric clash and electrostatic repulsion are indicated by the red lines. Bottom: Nucleotide exchange assay reveals a strict requirement in the lever segment for exchange of BODIPY-GDP. (c) Calculated electrostatic surface potential map (unit k B T/e) shows high negative charge in the OtDUB GEF lever formed by a tri-carbonyl motif at the vicinity of the diphosphate group of GDP.

    Article Snippet: BODIPY-GDP nucleotide exchange assays GTPases at a final concentration of 10 μM were loaded with sub-stoichiometric (1/4 [GTPase]) concentrations of GDP-BODIPY-FL (Thermo Scientific) in the presence of 50 mM Tris pH 7.5, 50 mM NaCl, 1 mM DTT, supplemented with 2 mM EDTA to promote unloading of co-purified nucleotide.

    Techniques: Binding 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

    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