alexa fluor Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher alexa fluor
    Figure 5. Convergence of the β1 integrin endocytic pathway and the autophagic pathway in migrating cells during starvation. HeLa-GFP-LC3 cells were stained with anti-β1 integrin antibody (P5D2 1:50) and <t>Alexa</t> fluor 594 <t>anti-IgG</t>
    Alexa Fluor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22435 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor/product/Thermo Fisher
    Average 99 stars, based on 22435 article reviews
    Price from $9.99 to $1999.99
    alexa fluor - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher alexa fluo 488 green
    Mouse primary and immortalized osteoblast cell proliferation. Proliferation of primary and immortalized cells was immunostained using a mouse monoclonal anti-BrdU antibody (1:100 dilution) after a 4-h BrdU incorporation (30 μM), followed by a 1:1,000 dilution of the secondary antibody (goat-anti-mouse) with <t>Alexa</t> Fluo® 488 green. For nucleus staining, the cells were incubated with a 1:5,000 dilution of Hoechst. Images were obtained by a Nikon inverted microscope and proliferative cells were expressed as a percentage of the number of BrdU positive cells relative to the total number of Hoechst positive nuclei. BrdU positive staining of the primary cells acts as 100% and asterisk (*) shows significant difference between the primary and immortalized cells ( p
    Alexa Fluo 488 Green, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluo 488 green/product/Thermo Fisher
    Average 99 stars, based on 29 article reviews
    Price from $9.99 to $1999.99
    alexa fluo 488 green - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher alexa fluoro 488 fluorophore
    Mouse primary and immortalized osteoblast cell proliferation. Proliferation of primary and immortalized cells was immunostained using a mouse monoclonal anti-BrdU antibody (1:100 dilution) after a 4-h BrdU incorporation (30 μM), followed by a 1:1,000 dilution of the secondary antibody (goat-anti-mouse) with <t>Alexa</t> Fluo® 488 green. For nucleus staining, the cells were incubated with a 1:5,000 dilution of Hoechst. Images were obtained by a Nikon inverted microscope and proliferative cells were expressed as a percentage of the number of BrdU positive cells relative to the total number of Hoechst positive nuclei. BrdU positive staining of the primary cells acts as 100% and asterisk (*) shows significant difference between the primary and immortalized cells ( p
    Alexa Fluoro 488 Fluorophore, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluoro 488 fluorophore/product/Thermo Fisher
    Average 99 stars, based on 78 article reviews
    Price from $9.99 to $1999.99
    alexa fluoro 488 fluorophore - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher alexa fluor 647
    Colocalization of MOR and SSTR2. (A) The distribution of SSTR2 (magenta, detected with Atto 488) and MOR (cyan, detected with <t>Alexa</t> Fluor 647) was determined in a region of normal epithelial pancreatic cells. Scale bar, 2 μm. Peak centers are shown. (B) The distribution of SSTR2 (magenta, detected with Atto 488) and MOR (cyan, detected with Alexa Fluor 647) was determined in a region of PANC-1 cells. Scale bar, 2 μm. Overlap is evident in dark blue. Peak centers are shown. (C) Cross-correlation curves with SEM show colocalization between MOR and SSTR2 in both PANC-1 cells (gray diamonds, 41 regions from 22 cells) and PANC-1 MCTS (red triangles, 40 regions from 21 cells). In contrast, colocalization was not observed in normal pancreatic epithelial cells (blue circles, 50 regions from 21 cells). These results represent combined data obtained using two labeling schemes: 1) MOR detected with Alexa <t>Fluor</t> 647 and SSTR2 detected with Atto 488 and, 2) MOR detected with Atto 488 and SSTR2 detected with Alexa Fluor 647. Individual curves are given in Supplemental Figure S6. In all cases, no long-range correlations were observed. (D) Cell lysates from either normal pancreatic epithelial cells or PANC-1 cells were immunoprecipitated with anti-MOR antibody and immunoblotted with anti-SSTR2 antibody. SSTR2 was detected in the MOR immunoprecipitate in the PANC-1 cell line.
    Alexa Fluor 647, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12035 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 647/product/Thermo Fisher
    Average 99 stars, based on 12035 article reviews
    Price from $9.99 to $1999.99
    alexa fluor 647 - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    94
    Thermo Fisher alexa 647 fluor
    Seldegs increase the accumulation of antigen-specific antibodies in human endothelial (HMEC-1) cells expressing FcRn-GFP. ( a ) HMEC-1 cells were incubated with 100 nM Alexa 647-labelled 8-18C5 (MOG-specific) or TZB (HER2-specific) in complex with 400 nM MOG-Seldeg/MOG-WT or HER2-Seldeg/HER2-WT for 30 min and chased for 0 (30' P) or 60 min (30' P, 60' C). Mean fluorescence intensities (MFI) of Alexa 647-labelled 8-18C5 or TZB for triplicate samples were determined by flow cytometry. Error bars indicate s.d. ( b , c ) HMEC-1 cells were plated on coverslips and treated as in a , except that Seldegs or control WT proteins were labelled with Alexa 555 and cells were fixed for microscopy. Images of representative cells from multiple cells analysed are shown with GFP, Alexa 555 and <t>Alexa</t> 647 in overlays pseudocoloured green, red and blue, respectively. Representative endosomes in the insets are cropped and expanded. ( d ) HMEC-1 cells were pre-pulsed with Alexa 555-labelled dextran for 2 h, washed and pulsed with 8-18C5 in complex with MOG-Seldeg, MOG-WT and HER2-Seldeg (concentrations and labels as for a ) for 30 min, followed by an 8 h chase. Cells were washed, fixed and imaged, and images for a representative cell from multiple cells analysed are presented. Representative lysosomes in the insets are cropped and expanded. For the overlay, GFP, Alexa 555 and Alexa 647 are pseudocoloured as in b . For b – d , scale bars=5 μm, and for insets, scale bars=0.25 μm. Data shown are representative of at least two independent experiments.
    Alexa 647 Fluor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa 647 fluor/product/Thermo Fisher
    Average 94 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    alexa 647 fluor - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    99
    Thermo Fisher alexa 488 fluor
    Acid-switching results in increased accumulation of ADCs in HER2-expressing tumor cells. Cells were incubated with 10 nM Alexa 488-labeled MMAE-conjugated antibody (WT, SG, YS or control hen egg lysozyme-specific antibody, C) for the indicated times at 37 °C, washed, incubated with 5 μg/ml Alexa 488-specific antibody and analyzed by flow cytometry. Mean fluorescence intensities (mean values of independent triplicate cell samples) for <t>Alexa</t> 488 fluorescence are shown. Error bars indicate SD. Statistically significant differences are indicated by * (unpaired two-tailed t -test). Two independent experiments were carried out with similar results.
    Alexa 488 Fluor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa 488 fluor/product/Thermo Fisher
    Average 99 stars, based on 86 article reviews
    Price from $9.99 to $1999.99
    alexa 488 fluor - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    97
    Thermo Fisher alexa fluor 405
    Neuronal calcium sensor (NCS1) in the Golgi apparatus recognizes Ca 2+ signal for focal release of mannosidase-II vesicles. A, mCherrry-MAN2A-expressing U937 macrophages were incubated with latex beads (30 min) or infected with PKH67-labeled H37Rv (1 h). Cells were fixed and stained with anti-NCS1 antibody followed by <t>Alexa</t> Fluor 405-labeled secondary antibody. For the upper panel, latex beads were given green pseudo-color using Imaris ( scale bar, 4 μm). B, upper panel , in U937 macrophages, incubated with beads for 30 min or 1 h, samples were stained with anti-NCS1 antibody. Presence of NCS1 at the bead surface was calculated using the 3D spot creation module in Imaris 7.2 software. The box plot at right shows data from more than 100 beads from two independent experiments. For lower panel , mCherry-MAN2A ( red )-expressing U937-derived macrophages were infected with PKH67-labeled H37Rv ( green ) for 1 and 2 h. At the respective time points, samples were fixed and stained with anti-NCS1 antibody followed by Alexa Fluor 405-tagged secondary antibody. The images are representative of the 1-h time point. Percent co-localization of H37Rv with mannosidase-II, NCS1, or both mannosidase-II and NCS1 was calculated using Imaris 7.2. The data represent an average of more than 150 bacteria from three different experiments (values ± S.D.). DIC , differential interference contrast. C, co-localization of mannosidase-II and NCS1 in U937 macrophages. U937 macrophages expressing mCherry-MAN2A were stained with anti-NCS1 antibody followed by secondary antibody (pseudo-colored green ) ( scale bar, 15 μm). White box in the merge panel identifies the area that was magnified for the zoom panel. D, siRNA-mediated knockdown of NCS1 was confirmed by Western blottings on the whole cell lysates from the transfected cells. Knockdown was monitored at 48 h post-transfection. E, THP-1-derived macrophages treated with NCS1 siRNA were incubated with GFP expressing E. coli for 5 and 15 min. Cells were stained with anti-mannosidase-II antibody to assess the recruitment of mannosidase-II at the phagosomes in NCS1-depleted cells. Percent co-localization of E. coli with mannosidase-II was calculated using Imaris 7.2. The data represent an average of more than 150 bacteria from three different experiments (values ± S.E., *, p value
    Alexa Fluor 405, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 810 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 405/product/Thermo Fisher
    Average 97 stars, based on 810 article reviews
    Price from $9.99 to $1999.99
    alexa fluor 405 - by Bioz Stars, 2020-08
    97/100 stars
      Buy from Supplier

    99
    Thermo Fisher alexa fluor 488
    Myo1b is associated with and propagates with actin waves. (A) Merged fluorescence images acquired with SIM of an actin wave costained with anti-Myo1b antibodies, <t>Alexa</t> <t>Fluor</t> 488 phalloidin, and anti–β3-tubulin antibody and images of the 2.5× enlargement of the region marked by a white box are shown. (B) The propagation of F-actin structures and mCherry-Myo1b has been analyzed in cortical neurons expressing LifeAct-EGFP and mCherry-Myo1b at DIV1 by time-lapse spinning confocal microscopy. The first frame of Video 1 (one frame per minute) for LifeAct-EGFP and mCherry-Myo1b and the merged kymographs of 36 frames for the region marked on the first frame by a white lane for the two recombinant proteins are shown. The increase of fluorescence intensity for LifeAct-EGFP correlated with the increase of fluorescence intensity for mCherry-Myo1b. (C) The propagation of PIP3 and mCherry-Myo1b has been analyzed in cortical neurons expressing AKT-PH-EGFP and mCherry-Myo1b at DIV1 by time-lapse spinning confocal microscopy. The first frame of Video 2 and kymographs of 36 frames for the region marked on the first frame by a white lane for the two recombinant proteins are shown. The increase of fluorescence intensity for AKT-PH-EGFP correlated with the increase of fluorescence intensity for mCherry-Myo1b. Bars: (A) 10 µm; (B and C) 20 µm. T, time
    Alexa Fluor 488, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 71255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 488/product/Thermo Fisher
    Average 99 stars, based on 71255 article reviews
    Price from $9.99 to $1999.99
    alexa fluor 488 - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    97
    Thermo Fisher alexa fluor 546
    Panel A shows confocal images demonstrating membrane binding (arrows) and internalization (arrow heads) of <t>Alexa</t> Fluor 546 (A546) labeled TCRm RL6A by hCMEC/D3 cells (red fluorescence signal). Cells were paraformaldehyde-fixed after 15 min incubation with the antibodies at 37°C. No staining was detectable with an A546 labeled isotype control antibody. In this panel and all other panels nuclei were stained with the far-red DNA dye DRAQ5 and pseudo-colored in blue. DIC = differential interference contrast. Panel B indicates that the TCRm RL6A and the pan HLA-A2 antibody BB7.2 (both A546 labeled, red fluorescence signal) were internalized into endosomal compartments: the arrows and arrowheads in the top group of confocal images highlight colocalization of RL6A or BB7.2, respectively, with the early endosomal marker EEA1 labeled by Alexa Fluor 488 (A488, green fluorescence signal), as evident from the merged images (yellow color). In contrast, the bottom group of images shows lack of colocalization with caveolin 1 (CAV1) by labeling of distinct structures, which do not overlay in the merged images. The hCMEC/D3 cells were incubated with A546 labeled antibodies RL6A or BB7.2 for 30 min at 37°C, followed by fixation and staining for EEA1 or CAV1 (both detected by A488 labeled secondary antibodies). Panel C depicts images of live hCMEC/D3 cells after 30 min of incubation with A546-labeled RL6A, BB7.2 or isotype control antibody. Both RL6A and BB7.2 bind to the cell membrane (arrows) and undergo internalization (arrowheads), while the isotype control showed no interaction with the cells. Panel D compares cellular uptake of RL6A after pre-incubation of the antibody with either an irrelevant peptide/A2 complex in solution (denoted KLM tetramer) or with the cognate YLL/A2 complex (YLL tetramer). Uptake is virtually abolished by YLL tetramer, but unaffected by the irrelevant epitope. The Z-stack provides further evidence for the internalization process of the labeled TCRm (here after 30 min of incubation with the cells). The arrowhead highlights one of the vesicles with clear intracellular localization. Scale bars in all panels = 5 μm.
    Alexa Fluor 546, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 5720 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 546/product/Thermo Fisher
    Average 97 stars, based on 5720 article reviews
    Price from $9.99 to $1999.99
    alexa fluor 546 - by Bioz Stars, 2020-08
    97/100 stars
      Buy from Supplier

    99
    Thermo Fisher alexa fluor 594
    Generation of antioxidant over-expressing parasites. Transgenic parasites were created in the RHΔhxgprt background strain using plasmids designed to over-express HA-tagged versions of glutaredoxin (Glut), catalase (Cat), or peroxiredoxin 2 (PRx2) under the control of the gra1 gene promoter. ( a ) Over-expression was verified using quantitative real-time PCR to measure transcript levels. ( b ) Western blot analysis was used to confirm protein production and that the resultant proteins were the appropriate size. ( c ) Immunofluorescence assays were performed for protein localization, utilizing a rabbit monoclonal antibody against the HA tag and an <t>Alexa</t> <t>Fluor</t> 594 secondary antibody. ( d ) General parasite replication was determined by doubling assay. Confluent HFF in 24-well plates were infected with the indicated parasite lines for 5 hours before the medium was replaced with fresh growth medium. Parasites were allowed to grow for 32 hours before being methanol-fixed and stained with DiffQuick. The number of individual parasites per vacuole was enumerated for at least 50 vacuoles. Data is presented as percent of vacuoles from 4 experiments. Student’s t-test was employed for statistical analysis. Scale bars equal 2 μm.
    Alexa Fluor 594, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16941 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 594/product/Thermo Fisher
    Average 99 stars, based on 16941 article reviews
    Price from $9.99 to $1999.99
    alexa fluor 594 - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    96
    Thermo Fisher alexa fluor 647 dye
    Characterization of VCAM-1 + macrophages in the CHT. a, Transgenic Tg(kdrl:eGFP) embryos, stained with an anti-VCAM-1 antibody (magenta, arrows), show that the VCAM-1 + macrophage first appeared in the CHT at 32 h.p.f. b, Tg(kdrl:eGFP) embryos in the vcam1 cas011 , itga4 cas005 or runx1 w84x mutant background are stained with an anti-VCAM-1 antibody (magenta, white arrows) at 54 h.p.f. Signals in itga4 cas005 and runx1 w84x are similar to that in wild-type siblings, whereas there is almost no detectable signal in vcam1 cas011 mutants. c, Schematic diagrams (left) and confocal imaging (right) of VCAM-1 + macrophages (labelled with <t>Alexa</t> <t>Fluor</t> 647 dye-conjugated anti-VCAM-1 antibody by intravascular injection) that patrol the CHT of wild-type embryos. VCAM-1 + macrophages were mainly located intravascularly ( > 91%) with round or unpolarized cell morphology ( > 84%). Cross indicates the original position of VCAM-1 + . DA, dorsal aorta; VC, venous capillaries. Scale bars, 50 μm ( a, b ) and 20 μm ( c ).
    Alexa Fluor 647 Dye, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 329 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 647 dye/product/Thermo Fisher
    Average 96 stars, based on 329 article reviews
    Price from $9.99 to $1999.99
    alexa fluor 647 dye - by Bioz Stars, 2020-08
    96/100 stars
      Buy from Supplier

    99
    Thermo Fisher alexa fluor 546 alexa
    Characterization of VCAM-1 + macrophages in the CHT. a, Transgenic Tg(kdrl:eGFP) embryos, stained with an anti-VCAM-1 antibody (magenta, arrows), show that the VCAM-1 + macrophage first appeared in the CHT at 32 h.p.f. b, Tg(kdrl:eGFP) embryos in the vcam1 cas011 , itga4 cas005 or runx1 w84x mutant background are stained with an anti-VCAM-1 antibody (magenta, white arrows) at 54 h.p.f. Signals in itga4 cas005 and runx1 w84x are similar to that in wild-type siblings, whereas there is almost no detectable signal in vcam1 cas011 mutants. c, Schematic diagrams (left) and confocal imaging (right) of VCAM-1 + macrophages (labelled with <t>Alexa</t> <t>Fluor</t> 647 dye-conjugated anti-VCAM-1 antibody by intravascular injection) that patrol the CHT of wild-type embryos. VCAM-1 + macrophages were mainly located intravascularly ( > 91%) with round or unpolarized cell morphology ( > 84%). Cross indicates the original position of VCAM-1 + . DA, dorsal aorta; VC, venous capillaries. Scale bars, 50 μm ( a, b ) and 20 μm ( c ).
    Alexa Fluor 546 Alexa, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 546 alexa/product/Thermo Fisher
    Average 99 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    alexa fluor 546 alexa - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    Image Search Results


    Figure 5. Convergence of the β1 integrin endocytic pathway and the autophagic pathway in migrating cells during starvation. HeLa-GFP-LC3 cells were stained with anti-β1 integrin antibody (P5D2 1:50) and Alexa fluor 594 anti-IgG

    Journal: Cell Cycle

    Article Title: Autophagy modulates cell migration and β1 integrin membrane recycling

    doi: 10.4161/cc.26298

    Figure Lengend Snippet: Figure 5. Convergence of the β1 integrin endocytic pathway and the autophagic pathway in migrating cells during starvation. HeLa-GFP-LC3 cells were stained with anti-β1 integrin antibody (P5D2 1:50) and Alexa fluor 594 anti-IgG

    Article Snippet: Alexa Fluor 555- and 594-conjugated anti-IgG antibodies (ref: and ) and Lysotracker D99 were from Molecular Probes, Invitrogen Co.

    Techniques: Staining

    Mouse primary and immortalized osteoblast cell proliferation. Proliferation of primary and immortalized cells was immunostained using a mouse monoclonal anti-BrdU antibody (1:100 dilution) after a 4-h BrdU incorporation (30 μM), followed by a 1:1,000 dilution of the secondary antibody (goat-anti-mouse) with Alexa Fluo® 488 green. For nucleus staining, the cells were incubated with a 1:5,000 dilution of Hoechst. Images were obtained by a Nikon inverted microscope and proliferative cells were expressed as a percentage of the number of BrdU positive cells relative to the total number of Hoechst positive nuclei. BrdU positive staining of the primary cells acts as 100% and asterisk (*) shows significant difference between the primary and immortalized cells ( p

    Journal: Cell and tissue research

    Article Title: Development and characterization of a mouse floxed Bmp2 osteoblast cell line that retains osteoblast genotype and phenotype

    doi: 10.1007/s00441-010-1120-3

    Figure Lengend Snippet: Mouse primary and immortalized osteoblast cell proliferation. Proliferation of primary and immortalized cells was immunostained using a mouse monoclonal anti-BrdU antibody (1:100 dilution) after a 4-h BrdU incorporation (30 μM), followed by a 1:1,000 dilution of the secondary antibody (goat-anti-mouse) with Alexa Fluo® 488 green. For nucleus staining, the cells were incubated with a 1:5,000 dilution of Hoechst. Images were obtained by a Nikon inverted microscope and proliferative cells were expressed as a percentage of the number of BrdU positive cells relative to the total number of Hoechst positive nuclei. BrdU positive staining of the primary cells acts as 100% and asterisk (*) shows significant difference between the primary and immortalized cells ( p

    Article Snippet: The cells were then incubated with a mouse monoclonal anti-BrdU antibody (1:100; Santa Cruz Biotechnology), followed by a 1:1,000 dilution of the secondary antibodies (goat-anti-mouse) with Alexa Fluo® 488 green (Molecular Probes).

    Techniques: BrdU Incorporation Assay, Staining, Incubation, Inverted Microscopy

    Colocalization of MOR and SSTR2. (A) The distribution of SSTR2 (magenta, detected with Atto 488) and MOR (cyan, detected with Alexa Fluor 647) was determined in a region of normal epithelial pancreatic cells. Scale bar, 2 μm. Peak centers are shown. (B) The distribution of SSTR2 (magenta, detected with Atto 488) and MOR (cyan, detected with Alexa Fluor 647) was determined in a region of PANC-1 cells. Scale bar, 2 μm. Overlap is evident in dark blue. Peak centers are shown. (C) Cross-correlation curves with SEM show colocalization between MOR and SSTR2 in both PANC-1 cells (gray diamonds, 41 regions from 22 cells) and PANC-1 MCTS (red triangles, 40 regions from 21 cells). In contrast, colocalization was not observed in normal pancreatic epithelial cells (blue circles, 50 regions from 21 cells). These results represent combined data obtained using two labeling schemes: 1) MOR detected with Alexa Fluor 647 and SSTR2 detected with Atto 488 and, 2) MOR detected with Atto 488 and SSTR2 detected with Alexa Fluor 647. Individual curves are given in Supplemental Figure S6. In all cases, no long-range correlations were observed. (D) Cell lysates from either normal pancreatic epithelial cells or PANC-1 cells were immunoprecipitated with anti-MOR antibody and immunoblotted with anti-SSTR2 antibody. SSTR2 was detected in the MOR immunoprecipitate in the PANC-1 cell line.

    Journal: Molecular Biology of the Cell

    Article Title: Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

    doi: 10.1091/mbc.E16-06-0427

    Figure Lengend Snippet: Colocalization of MOR and SSTR2. (A) The distribution of SSTR2 (magenta, detected with Atto 488) and MOR (cyan, detected with Alexa Fluor 647) was determined in a region of normal epithelial pancreatic cells. Scale bar, 2 μm. Peak centers are shown. (B) The distribution of SSTR2 (magenta, detected with Atto 488) and MOR (cyan, detected with Alexa Fluor 647) was determined in a region of PANC-1 cells. Scale bar, 2 μm. Overlap is evident in dark blue. Peak centers are shown. (C) Cross-correlation curves with SEM show colocalization between MOR and SSTR2 in both PANC-1 cells (gray diamonds, 41 regions from 22 cells) and PANC-1 MCTS (red triangles, 40 regions from 21 cells). In contrast, colocalization was not observed in normal pancreatic epithelial cells (blue circles, 50 regions from 21 cells). These results represent combined data obtained using two labeling schemes: 1) MOR detected with Alexa Fluor 647 and SSTR2 detected with Atto 488 and, 2) MOR detected with Atto 488 and SSTR2 detected with Alexa Fluor 647. Individual curves are given in Supplemental Figure S6. In all cases, no long-range correlations were observed. (D) Cell lysates from either normal pancreatic epithelial cells or PANC-1 cells were immunoprecipitated with anti-MOR antibody and immunoblotted with anti-SSTR2 antibody. SSTR2 was detected in the MOR immunoprecipitate in the PANC-1 cell line.

    Article Snippet: Secondary antibodies were labeled with Alexa Fluor 405, Alexa Fluor 647 (Life Technologies), or Atto 488 (Sigma-Aldrich) containing an N -hydroxysuccinidimidyl ester (NHS) group for conjugation to proteins.

    Techniques: Labeling, Immunoprecipitation

    Pulse labelling of nascent DNA in E. coli cells with EdU. ( a ) Pulse labelling experiment in a fast-growing E. coli batch culture (LB medium, 32 °C). Growth curves of cultures with and without EdU are shown in the upper left panel. Culture aliquots were fixed after 2, 3.5 and 5 min and labelled with Alexa Fluor 647 via click-chemistry. Parallel multi-colour imaging was performed as described in the Methods section. Nucleoids were probed using JF 503 ). Increasing EdU incubation time leads to an increase of nucleoid coverage (ratio of the area populated by pulse-labelled DNA and the entire nucleoid) (upper right panel). The lower panel shows representative cells for the indicated EdU pulse durations, labelled for nascent DNA (red hot), the entire nucleoid (cyan hot) and the membrane (green). Outlines were determined in the membrane PAINT image and are shown by dashed lines in the DNA d STORM and PAINT images. ( b ) Pulse labelling during metabolic arrest. MG1655 cells were grown in LB medium at 32 °C. Growth curves of cultures with and without SHX are shown in the upper left panel. The treated culture was split into 3 cultures and EdU was added for the last 30 min of SHX treatment, finally resulting in cells arrested for 30 min, 60 min and 90 min. Parallel multi-colour imaging was performed as described in the Methods section. The nucleoid coverage of nascent DNA decreases with the time of SHX treatment (upper right panel). The lower panel shows representative cells for the different time points during metabolic arrest, the colour-coding is kept identical to ( a ). Bars represent mean values and error bars the respecting standard deviation (scale bars: 1 µm).

    Journal: Scientific Reports

    Article Title: A toolbox for multiplexed super-resolution imaging of the E. coli nucleoid and membrane using novel PAINT labels

    doi: 10.1038/s41598-018-33052-3

    Figure Lengend Snippet: Pulse labelling of nascent DNA in E. coli cells with EdU. ( a ) Pulse labelling experiment in a fast-growing E. coli batch culture (LB medium, 32 °C). Growth curves of cultures with and without EdU are shown in the upper left panel. Culture aliquots were fixed after 2, 3.5 and 5 min and labelled with Alexa Fluor 647 via click-chemistry. Parallel multi-colour imaging was performed as described in the Methods section. Nucleoids were probed using JF 503 ). Increasing EdU incubation time leads to an increase of nucleoid coverage (ratio of the area populated by pulse-labelled DNA and the entire nucleoid) (upper right panel). The lower panel shows representative cells for the indicated EdU pulse durations, labelled for nascent DNA (red hot), the entire nucleoid (cyan hot) and the membrane (green). Outlines were determined in the membrane PAINT image and are shown by dashed lines in the DNA d STORM and PAINT images. ( b ) Pulse labelling during metabolic arrest. MG1655 cells were grown in LB medium at 32 °C. Growth curves of cultures with and without SHX are shown in the upper left panel. The treated culture was split into 3 cultures and EdU was added for the last 30 min of SHX treatment, finally resulting in cells arrested for 30 min, 60 min and 90 min. Parallel multi-colour imaging was performed as described in the Methods section. The nucleoid coverage of nascent DNA decreases with the time of SHX treatment (upper right panel). The lower panel shows representative cells for the different time points during metabolic arrest, the colour-coding is kept identical to ( a ). Bars represent mean values and error bars the respecting standard deviation (scale bars: 1 µm).

    Article Snippet: The DNA of the EdU treated bacteria was labelled with Alexa Fluor 647 azide (Thermo Fisher Scientific) via click chemistry as described previously .

    Techniques: Imaging, Incubation, Standard Deviation

    Annexin-V/Alexa Fluor™ 647 and Propidium iodide stained U87-MG cells post treatment with free and nano GSK (5 µM). Population of cells that are in early apoptosis, late apoptosis and necrosis at 24, 48 and 72 h are mentioned. The first row indicates cells that received no treatment.

    Journal: Bioengineering

    Article Title: GSK461364A, a Polo-Like Kinase-1 Inhibitor Encapsulated in Polymeric Nanoparticles for the Treatment of Glioblastoma Multiforme (GBM)

    doi: 10.3390/bioengineering5040083

    Figure Lengend Snippet: Annexin-V/Alexa Fluor™ 647 and Propidium iodide stained U87-MG cells post treatment with free and nano GSK (5 µM). Population of cells that are in early apoptosis, late apoptosis and necrosis at 24, 48 and 72 h are mentioned. The first row indicates cells that received no treatment.

    Article Snippet: Annexin-V, Alexa Fluor™ 647 conjugate was purchased from Invitrogen™.

    Techniques: Staining

    Seldegs increase the accumulation of antigen-specific antibodies in human endothelial (HMEC-1) cells expressing FcRn-GFP. ( a ) HMEC-1 cells were incubated with 100 nM Alexa 647-labelled 8-18C5 (MOG-specific) or TZB (HER2-specific) in complex with 400 nM MOG-Seldeg/MOG-WT or HER2-Seldeg/HER2-WT for 30 min and chased for 0 (30' P) or 60 min (30' P, 60' C). Mean fluorescence intensities (MFI) of Alexa 647-labelled 8-18C5 or TZB for triplicate samples were determined by flow cytometry. Error bars indicate s.d. ( b , c ) HMEC-1 cells were plated on coverslips and treated as in a , except that Seldegs or control WT proteins were labelled with Alexa 555 and cells were fixed for microscopy. Images of representative cells from multiple cells analysed are shown with GFP, Alexa 555 and Alexa 647 in overlays pseudocoloured green, red and blue, respectively. Representative endosomes in the insets are cropped and expanded. ( d ) HMEC-1 cells were pre-pulsed with Alexa 555-labelled dextran for 2 h, washed and pulsed with 8-18C5 in complex with MOG-Seldeg, MOG-WT and HER2-Seldeg (concentrations and labels as for a ) for 30 min, followed by an 8 h chase. Cells were washed, fixed and imaged, and images for a representative cell from multiple cells analysed are presented. Representative lysosomes in the insets are cropped and expanded. For the overlay, GFP, Alexa 555 and Alexa 647 are pseudocoloured as in b . For b – d , scale bars=5 μm, and for insets, scale bars=0.25 μm. Data shown are representative of at least two independent experiments.

    Journal: Nature Communications

    Article Title: Engineered clearing agents for the selective depletion of antigen-specific antibodies

    doi: 10.1038/ncomms15314

    Figure Lengend Snippet: Seldegs increase the accumulation of antigen-specific antibodies in human endothelial (HMEC-1) cells expressing FcRn-GFP. ( a ) HMEC-1 cells were incubated with 100 nM Alexa 647-labelled 8-18C5 (MOG-specific) or TZB (HER2-specific) in complex with 400 nM MOG-Seldeg/MOG-WT or HER2-Seldeg/HER2-WT for 30 min and chased for 0 (30' P) or 60 min (30' P, 60' C). Mean fluorescence intensities (MFI) of Alexa 647-labelled 8-18C5 or TZB for triplicate samples were determined by flow cytometry. Error bars indicate s.d. ( b , c ) HMEC-1 cells were plated on coverslips and treated as in a , except that Seldegs or control WT proteins were labelled with Alexa 555 and cells were fixed for microscopy. Images of representative cells from multiple cells analysed are shown with GFP, Alexa 555 and Alexa 647 in overlays pseudocoloured green, red and blue, respectively. Representative endosomes in the insets are cropped and expanded. ( d ) HMEC-1 cells were pre-pulsed with Alexa 555-labelled dextran for 2 h, washed and pulsed with 8-18C5 in complex with MOG-Seldeg, MOG-WT and HER2-Seldeg (concentrations and labels as for a ) for 30 min, followed by an 8 h chase. Cells were washed, fixed and imaged, and images for a representative cell from multiple cells analysed are presented. Representative lysosomes in the insets are cropped and expanded. For the overlay, GFP, Alexa 555 and Alexa 647 are pseudocoloured as in b . For b – d , scale bars=5 μm, and for insets, scale bars=0.25 μm. Data shown are representative of at least two independent experiments.

    Article Snippet: 8-18C5 and TZB were fluorescently labelled with Alexa 647 Fluor (ThermoFisher Scientific) with antibody:dye ratios of 1.6 and 3, respectively.

    Techniques: Expressing, Incubation, Fluorescence, Flow Cytometry, Cytometry, Microscopy

    Fluorescent labeling of a natural RNA sample. A total RNA isolate from E. coli was reacted with 2′-N 3 -2′-dUTP and yeast PAP, and further subjected to CuAAC with Alexa Fluor 647 alkyne. Analysis by 8% denaturing PAGE. SYBR Gold scan (left panel), Alexa Fluor 647 scan (middle panel) and overlay (green: radioactivity; magenta: fluorescence; white: both; right panel) are given. The two main bands represent 23S and 16S ribosomal RNA.

    Journal: Nucleic Acids Research

    Article Title: Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry

    doi: 10.1093/nar/gks062

    Figure Lengend Snippet: Fluorescent labeling of a natural RNA sample. A total RNA isolate from E. coli was reacted with 2′-N 3 -2′-dUTP and yeast PAP, and further subjected to CuAAC with Alexa Fluor 647 alkyne. Analysis by 8% denaturing PAGE. SYBR Gold scan (left panel), Alexa Fluor 647 scan (middle panel) and overlay (green: radioactivity; magenta: fluorescence; white: both; right panel) are given. The two main bands represent 23S and 16S ribosomal RNA.

    Article Snippet: Alexa Fluor 647 alkyne, Alexa Fluor 488 alkyne and biotin-alkyne were purchased from Invitrogen.

    Techniques: Labeling, Polyacrylamide Gel Electrophoresis, Radioactivity, Fluorescence

    Creating an internal fluorescent label. ( A ) General reaction scheme for creation of terminal (upper right corner) or internal modifications (lower right corner). An internal modification can be created by first adding an N 3 -modified nucleotide to the 3′-terminus of the RNA sequence, connecting this RNA to a second RNA sequence via splinted ligation, and subjecting the product, with an internal N 3 -modification to CuAAC. ( B ) Splinted ligation of RNA1 and RNA4, employing different ligases (RNL2 and DNL) under different reaction conditions (time and temperature). Analysis by 15% seqPAGE. ( C ) Addition of 2′-N 3 -guanosine to RNA1, followed by splinted ligation to RNA4 (using RNL2), and CuAAC with Alexa Fluor 488/647 alkyne, with or without the use of a helper DNA that forces the modified position into a 9-nt bulge loop. Analysis by 15% seqPAGE. Radioactivity scan (left panel), and an overlay of Alexa Fluor 488 scan (green, middle two lanes) and Alexa Fluor 647 scan (magenta, right two lanes) are shown. ( D ) Formation of 9 nt bulge loop to assist CuAAC. N.R.: no reaction control.

    Journal: Nucleic Acids Research

    Article Title: Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry

    doi: 10.1093/nar/gks062

    Figure Lengend Snippet: Creating an internal fluorescent label. ( A ) General reaction scheme for creation of terminal (upper right corner) or internal modifications (lower right corner). An internal modification can be created by first adding an N 3 -modified nucleotide to the 3′-terminus of the RNA sequence, connecting this RNA to a second RNA sequence via splinted ligation, and subjecting the product, with an internal N 3 -modification to CuAAC. ( B ) Splinted ligation of RNA1 and RNA4, employing different ligases (RNL2 and DNL) under different reaction conditions (time and temperature). Analysis by 15% seqPAGE. ( C ) Addition of 2′-N 3 -guanosine to RNA1, followed by splinted ligation to RNA4 (using RNL2), and CuAAC with Alexa Fluor 488/647 alkyne, with or without the use of a helper DNA that forces the modified position into a 9-nt bulge loop. Analysis by 15% seqPAGE. Radioactivity scan (left panel), and an overlay of Alexa Fluor 488 scan (green, middle two lanes) and Alexa Fluor 647 scan (magenta, right two lanes) are shown. ( D ) Formation of 9 nt bulge loop to assist CuAAC. N.R.: no reaction control.

    Article Snippet: Alexa Fluor 647 alkyne, Alexa Fluor 488 alkyne and biotin-alkyne were purchased from Invitrogen.

    Techniques: Modification, Sequencing, Ligation, Radioactivity

    Detection limit for RNA1 modified with each of the four 2′-N 3 -2′-dNTPs and conjugated with Alexa Fluor 647 alkyne. Samples (same as in Figure 3 A) were diluted and analyzed by 12% seqPAGE in amounts from 25 amol to 2.5 fmol. Fluorescence scan (Typhoon 9400, pixel size: 100 µm, PMT: 800 V, high sensitivity) is shown.

    Journal: Nucleic Acids Research

    Article Title: Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry

    doi: 10.1093/nar/gks062

    Figure Lengend Snippet: Detection limit for RNA1 modified with each of the four 2′-N 3 -2′-dNTPs and conjugated with Alexa Fluor 647 alkyne. Samples (same as in Figure 3 A) were diluted and analyzed by 12% seqPAGE in amounts from 25 amol to 2.5 fmol. Fluorescence scan (Typhoon 9400, pixel size: 100 µm, PMT: 800 V, high sensitivity) is shown.

    Article Snippet: Alexa Fluor 647 alkyne, Alexa Fluor 488 alkyne and biotin-alkyne were purchased from Invitrogen.

    Techniques: Modification, Fluorescence

    Fluorescent labeling of RNA by CuAAC or SPAAC. RNA1, modified with each of the four 2′-N 3 -2′-dNTPs under optimized conditions and further conjugated with fluorescent dyes. Analysis by 15% seqPAGE. Radioactivity scan (upper panel), fluorescence scan (middle panel) and an overlay of both (green: radioactivity; magenta: fluorescence; white: both; lower panel) are given. ( A ) Conjugation with Alexa Fluor 647 alkyne by CuAAC. ( B ) Conjugation with DIBAC Fluor 488 by SPAAC.

    Journal: Nucleic Acids Research

    Article Title: Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry

    doi: 10.1093/nar/gks062

    Figure Lengend Snippet: Fluorescent labeling of RNA by CuAAC or SPAAC. RNA1, modified with each of the four 2′-N 3 -2′-dNTPs under optimized conditions and further conjugated with fluorescent dyes. Analysis by 15% seqPAGE. Radioactivity scan (upper panel), fluorescence scan (middle panel) and an overlay of both (green: radioactivity; magenta: fluorescence; white: both; lower panel) are given. ( A ) Conjugation with Alexa Fluor 647 alkyne by CuAAC. ( B ) Conjugation with DIBAC Fluor 488 by SPAAC.

    Article Snippet: Alexa Fluor 647 alkyne, Alexa Fluor 488 alkyne and biotin-alkyne were purchased from Invitrogen.

    Techniques: Labeling, Modification, Radioactivity, Fluorescence, Conjugation Assay

    Acid-switching results in increased accumulation of ADCs in HER2-expressing tumor cells. Cells were incubated with 10 nM Alexa 488-labeled MMAE-conjugated antibody (WT, SG, YS or control hen egg lysozyme-specific antibody, C) for the indicated times at 37 °C, washed, incubated with 5 μg/ml Alexa 488-specific antibody and analyzed by flow cytometry. Mean fluorescence intensities (mean values of independent triplicate cell samples) for Alexa 488 fluorescence are shown. Error bars indicate SD. Statistically significant differences are indicated by * (unpaired two-tailed t -test). Two independent experiments were carried out with similar results.

    Journal: Nature biotechnology

    Article Title: Engineering a HER2-specific antibody-drug conjugate to increase lysosomal delivery and therapeutic efficacy

    doi: 10.1038/s41587-019-0073-7

    Figure Lengend Snippet: Acid-switching results in increased accumulation of ADCs in HER2-expressing tumor cells. Cells were incubated with 10 nM Alexa 488-labeled MMAE-conjugated antibody (WT, SG, YS or control hen egg lysozyme-specific antibody, C) for the indicated times at 37 °C, washed, incubated with 5 μg/ml Alexa 488-specific antibody and analyzed by flow cytometry. Mean fluorescence intensities (mean values of independent triplicate cell samples) for Alexa 488 fluorescence are shown. Error bars indicate SD. Statistically significant differences are indicated by * (unpaired two-tailed t -test). Two independent experiments were carried out with similar results.

    Article Snippet: ADCs were labeled with Alexa 488 Fluor (Thermo Fisher Scientific) or radiolabeled with 125 I-Iodogen [Perkin Elmer or MP Biomedical (Solon, OH, USA)] as described previously , except that a 1:1 molar ratio of Alexa 488:ADC was used during the labeling reaction.

    Techniques: Expressing, Incubation, Labeling, Flow Cytometry, Cytometry, Fluorescence, Two Tailed Test

    Neuronal calcium sensor (NCS1) in the Golgi apparatus recognizes Ca 2+ signal for focal release of mannosidase-II vesicles. A, mCherrry-MAN2A-expressing U937 macrophages were incubated with latex beads (30 min) or infected with PKH67-labeled H37Rv (1 h). Cells were fixed and stained with anti-NCS1 antibody followed by Alexa Fluor 405-labeled secondary antibody. For the upper panel, latex beads were given green pseudo-color using Imaris ( scale bar, 4 μm). B, upper panel , in U937 macrophages, incubated with beads for 30 min or 1 h, samples were stained with anti-NCS1 antibody. Presence of NCS1 at the bead surface was calculated using the 3D spot creation module in Imaris 7.2 software. The box plot at right shows data from more than 100 beads from two independent experiments. For lower panel , mCherry-MAN2A ( red )-expressing U937-derived macrophages were infected with PKH67-labeled H37Rv ( green ) for 1 and 2 h. At the respective time points, samples were fixed and stained with anti-NCS1 antibody followed by Alexa Fluor 405-tagged secondary antibody. The images are representative of the 1-h time point. Percent co-localization of H37Rv with mannosidase-II, NCS1, or both mannosidase-II and NCS1 was calculated using Imaris 7.2. The data represent an average of more than 150 bacteria from three different experiments (values ± S.D.). DIC , differential interference contrast. C, co-localization of mannosidase-II and NCS1 in U937 macrophages. U937 macrophages expressing mCherry-MAN2A were stained with anti-NCS1 antibody followed by secondary antibody (pseudo-colored green ) ( scale bar, 15 μm). White box in the merge panel identifies the area that was magnified for the zoom panel. D, siRNA-mediated knockdown of NCS1 was confirmed by Western blottings on the whole cell lysates from the transfected cells. Knockdown was monitored at 48 h post-transfection. E, THP-1-derived macrophages treated with NCS1 siRNA were incubated with GFP expressing E. coli for 5 and 15 min. Cells were stained with anti-mannosidase-II antibody to assess the recruitment of mannosidase-II at the phagosomes in NCS1-depleted cells. Percent co-localization of E. coli with mannosidase-II was calculated using Imaris 7.2. The data represent an average of more than 150 bacteria from three different experiments (values ± S.E., *, p value

    Journal: The Journal of Biological Chemistry

    Article Title: Ca2+-dependent Focal Exocytosis of Golgi-derived Vesicles Helps Phagocytic Uptake in Macrophages *

    doi: 10.1074/jbc.M116.743047

    Figure Lengend Snippet: Neuronal calcium sensor (NCS1) in the Golgi apparatus recognizes Ca 2+ signal for focal release of mannosidase-II vesicles. A, mCherrry-MAN2A-expressing U937 macrophages were incubated with latex beads (30 min) or infected with PKH67-labeled H37Rv (1 h). Cells were fixed and stained with anti-NCS1 antibody followed by Alexa Fluor 405-labeled secondary antibody. For the upper panel, latex beads were given green pseudo-color using Imaris ( scale bar, 4 μm). B, upper panel , in U937 macrophages, incubated with beads for 30 min or 1 h, samples were stained with anti-NCS1 antibody. Presence of NCS1 at the bead surface was calculated using the 3D spot creation module in Imaris 7.2 software. The box plot at right shows data from more than 100 beads from two independent experiments. For lower panel , mCherry-MAN2A ( red )-expressing U937-derived macrophages were infected with PKH67-labeled H37Rv ( green ) for 1 and 2 h. At the respective time points, samples were fixed and stained with anti-NCS1 antibody followed by Alexa Fluor 405-tagged secondary antibody. The images are representative of the 1-h time point. Percent co-localization of H37Rv with mannosidase-II, NCS1, or both mannosidase-II and NCS1 was calculated using Imaris 7.2. The data represent an average of more than 150 bacteria from three different experiments (values ± S.D.). DIC , differential interference contrast. C, co-localization of mannosidase-II and NCS1 in U937 macrophages. U937 macrophages expressing mCherry-MAN2A were stained with anti-NCS1 antibody followed by secondary antibody (pseudo-colored green ) ( scale bar, 15 μm). White box in the merge panel identifies the area that was magnified for the zoom panel. D, siRNA-mediated knockdown of NCS1 was confirmed by Western blottings on the whole cell lysates from the transfected cells. Knockdown was monitored at 48 h post-transfection. E, THP-1-derived macrophages treated with NCS1 siRNA were incubated with GFP expressing E. coli for 5 and 15 min. Cells were stained with anti-mannosidase-II antibody to assess the recruitment of mannosidase-II at the phagosomes in NCS1-depleted cells. Percent co-localization of E. coli with mannosidase-II was calculated using Imaris 7.2. The data represent an average of more than 150 bacteria from three different experiments (values ± S.E., *, p value

    Article Snippet: The secondary antibodies used in this study are Alexa Fluor 405 and Alexa Fluor 568 conjugates from Life Technologies, Inc.

    Techniques: Expressing, Incubation, Infection, Labeling, Staining, Software, Derivative Assay, Western Blot, Transfection

    Vesicles derived from Golgi apparatus are distinct from recycling endosome vesicles and are recruited independently. A, U937 cells expressing MAN2A-mCherry were fixed and stained with anti-VAMP3 antibody followed by Alexa Fluor 405-labeled secondary antibody ( scale bar, 15 μm). White box in the merge panel identifies the area that was magnified for the zoom panel. DIC , differential interference contrast. B, mCherry-MAN2A ( red )-expressing RAW264.7 macrophages were incubated with mouse serum-coated latex beads for 30 min or 1 h. At the respective time points, cells were immunostained with anti-VAMP-3 antibody followed by a secondary antibody tagged with Alexa Fluor 405 ( blue ). Presence of VAMP-3 or mannosidase-II at the bead surface was calculated using the 3D spot creation module in Imaris 7.2 software. The box plot at right shows data from more than 100 beads from two independent experiments ( scale bar, 5 μm). C, mCherry-MAN2A ( red )-expressing THP-1-derived macrophages were infected with PKH67-labeled H37Rv ( green ) for 1 and 2 h. At the respective time points, samples were fixed and stained with anti-VAMP-3 antibody followed by Alexa Fluor 405-tagged secondary antibody ( blue ). The images are representative of the 1-h time point. For the plots at right , % co-localization of H37Rv with mannosidase-II, VAMP-3, or both mannosidase-II and VAMP-3 was calculated using Imaris 7.2. The data represent an average of more than 150 bacteria from three different experiments (values ± S.D.; scale bar, 4 μm). D, U937 cells were incubated with serum-coated FITC-labeled latex beads in the presence or absence of BfA for 30 min. Samples were fixed and stained with anti-VAMP3 antibody followed by Alexa Fluor 405-labeled secondary antibody. Images were analyzed using 3D module in Imaris 7.2, and VAMP3 intensity distribution in the latex beads was calculated. E, U937 cells were incubated with GFP expressing E. coli in the presence or absence of BfA for 30 min. Samples were fixed and stained with anti-VAMP3 antibody followed by Alexa Fluor 405-labeled secondary antibody. Images were analyzed using 3D module in Imaris 7.2, and VAMP3 intensity distribution in the latex beads was calculated.

    Journal: The Journal of Biological Chemistry

    Article Title: Ca2+-dependent Focal Exocytosis of Golgi-derived Vesicles Helps Phagocytic Uptake in Macrophages *

    doi: 10.1074/jbc.M116.743047

    Figure Lengend Snippet: Vesicles derived from Golgi apparatus are distinct from recycling endosome vesicles and are recruited independently. A, U937 cells expressing MAN2A-mCherry were fixed and stained with anti-VAMP3 antibody followed by Alexa Fluor 405-labeled secondary antibody ( scale bar, 15 μm). White box in the merge panel identifies the area that was magnified for the zoom panel. DIC , differential interference contrast. B, mCherry-MAN2A ( red )-expressing RAW264.7 macrophages were incubated with mouse serum-coated latex beads for 30 min or 1 h. At the respective time points, cells were immunostained with anti-VAMP-3 antibody followed by a secondary antibody tagged with Alexa Fluor 405 ( blue ). Presence of VAMP-3 or mannosidase-II at the bead surface was calculated using the 3D spot creation module in Imaris 7.2 software. The box plot at right shows data from more than 100 beads from two independent experiments ( scale bar, 5 μm). C, mCherry-MAN2A ( red )-expressing THP-1-derived macrophages were infected with PKH67-labeled H37Rv ( green ) for 1 and 2 h. At the respective time points, samples were fixed and stained with anti-VAMP-3 antibody followed by Alexa Fluor 405-tagged secondary antibody ( blue ). The images are representative of the 1-h time point. For the plots at right , % co-localization of H37Rv with mannosidase-II, VAMP-3, or both mannosidase-II and VAMP-3 was calculated using Imaris 7.2. The data represent an average of more than 150 bacteria from three different experiments (values ± S.D.; scale bar, 4 μm). D, U937 cells were incubated with serum-coated FITC-labeled latex beads in the presence or absence of BfA for 30 min. Samples were fixed and stained with anti-VAMP3 antibody followed by Alexa Fluor 405-labeled secondary antibody. Images were analyzed using 3D module in Imaris 7.2, and VAMP3 intensity distribution in the latex beads was calculated. E, U937 cells were incubated with GFP expressing E. coli in the presence or absence of BfA for 30 min. Samples were fixed and stained with anti-VAMP3 antibody followed by Alexa Fluor 405-labeled secondary antibody. Images were analyzed using 3D module in Imaris 7.2, and VAMP3 intensity distribution in the latex beads was calculated.

    Article Snippet: The secondary antibodies used in this study are Alexa Fluor 405 and Alexa Fluor 568 conjugates from Life Technologies, Inc.

    Techniques: Derivative Assay, Expressing, Staining, Labeling, Incubation, Software, Infection

    Early recruitment of mannosidase-II at phagosomes and proteomic characterization. A, mCherry-MAN2A ( red )-expressing RAW264.7 macrophages were incubated with mouse serum-coated latex beads for 30 min or 1 h. At the respective time points, cells were immunostained with anti-TfR antibody followed by a secondary antibody tagged with Alexa Fluor 405 ( blue ). Presence of TfR or mannosidase-II at the bead surface was calculated in terms of fluorescence intensity using the 3D spot creation module in Imaris 7.2 software. The box plot at right shows data from more than 100 beads from two independent experiments ( scale bar, 5 μm). B, mCherry-MAN2A ( red )-expressing RAW264.7 macrophages were infected with PKH67-labeled H37Rv ( green ) for 1 and 2 h. At the respective time points, samples were fixed and stained with anti-transferrin receptor antibody followed by Alexa Fluor 405-tagged secondary antibody ( blue ). The images are representative of the 1-h time point. For the plots at right , % co-localization of H37Rv with mannosidase-II, TfR, or both mannosidase-II and TfR was calculated using Imaris 7.2. The data represent the average of more than 150 bacteria from three different experiments (values ± S.D.; scale bar, 5 μm). C, preparation of latex bead phagosomes from THP-1-derived macrophages on a sucrose density gradient (see “Materials and Methods”). D, THP-1-derived macrophages were incubated with latex beads (1 μm size) for 1 h. Phagosomes were isolated using differential density ultracentrifugation, and samples were probed for indicated markers using Western blottings. Molecular mass markers are the closest markers to the band of interest. WCL , whole cell lysate. E, latex beads phagosomes isolated from RAW264.7 macrophages were lysed and resolved on a 10% SDS-PAGE. The molecules below the 25-kDa molecular mass were cut from the gel and analyzed using mass spectrometry to identify enrichment of low molecular weight proteins (see “Materials and Methods”). The list of genes identified was then searched in the AMIGO2.0 database to establish functional association. Finally the representative network was constructed using Cytoscape 2.6.1. The pink nodes and edges in the network denote association with the Golgi apparatus.

    Journal: The Journal of Biological Chemistry

    Article Title: Ca2+-dependent Focal Exocytosis of Golgi-derived Vesicles Helps Phagocytic Uptake in Macrophages *

    doi: 10.1074/jbc.M116.743047

    Figure Lengend Snippet: Early recruitment of mannosidase-II at phagosomes and proteomic characterization. A, mCherry-MAN2A ( red )-expressing RAW264.7 macrophages were incubated with mouse serum-coated latex beads for 30 min or 1 h. At the respective time points, cells were immunostained with anti-TfR antibody followed by a secondary antibody tagged with Alexa Fluor 405 ( blue ). Presence of TfR or mannosidase-II at the bead surface was calculated in terms of fluorescence intensity using the 3D spot creation module in Imaris 7.2 software. The box plot at right shows data from more than 100 beads from two independent experiments ( scale bar, 5 μm). B, mCherry-MAN2A ( red )-expressing RAW264.7 macrophages were infected with PKH67-labeled H37Rv ( green ) for 1 and 2 h. At the respective time points, samples were fixed and stained with anti-transferrin receptor antibody followed by Alexa Fluor 405-tagged secondary antibody ( blue ). The images are representative of the 1-h time point. For the plots at right , % co-localization of H37Rv with mannosidase-II, TfR, or both mannosidase-II and TfR was calculated using Imaris 7.2. The data represent the average of more than 150 bacteria from three different experiments (values ± S.D.; scale bar, 5 μm). C, preparation of latex bead phagosomes from THP-1-derived macrophages on a sucrose density gradient (see “Materials and Methods”). D, THP-1-derived macrophages were incubated with latex beads (1 μm size) for 1 h. Phagosomes were isolated using differential density ultracentrifugation, and samples were probed for indicated markers using Western blottings. Molecular mass markers are the closest markers to the band of interest. WCL , whole cell lysate. E, latex beads phagosomes isolated from RAW264.7 macrophages were lysed and resolved on a 10% SDS-PAGE. The molecules below the 25-kDa molecular mass were cut from the gel and analyzed using mass spectrometry to identify enrichment of low molecular weight proteins (see “Materials and Methods”). The list of genes identified was then searched in the AMIGO2.0 database to establish functional association. Finally the representative network was constructed using Cytoscape 2.6.1. The pink nodes and edges in the network denote association with the Golgi apparatus.

    Article Snippet: The secondary antibodies used in this study are Alexa Fluor 405 and Alexa Fluor 568 conjugates from Life Technologies, Inc.

    Techniques: Expressing, Incubation, Fluorescence, Software, Infection, Labeling, Staining, Derivative Assay, Isolation, Western Blot, SDS Page, Mass Spectrometry, Molecular Weight, Functional Assay, Construct

    Myo1b is associated with and propagates with actin waves. (A) Merged fluorescence images acquired with SIM of an actin wave costained with anti-Myo1b antibodies, Alexa Fluor 488 phalloidin, and anti–β3-tubulin antibody and images of the 2.5× enlargement of the region marked by a white box are shown. (B) The propagation of F-actin structures and mCherry-Myo1b has been analyzed in cortical neurons expressing LifeAct-EGFP and mCherry-Myo1b at DIV1 by time-lapse spinning confocal microscopy. The first frame of Video 1 (one frame per minute) for LifeAct-EGFP and mCherry-Myo1b and the merged kymographs of 36 frames for the region marked on the first frame by a white lane for the two recombinant proteins are shown. The increase of fluorescence intensity for LifeAct-EGFP correlated with the increase of fluorescence intensity for mCherry-Myo1b. (C) The propagation of PIP3 and mCherry-Myo1b has been analyzed in cortical neurons expressing AKT-PH-EGFP and mCherry-Myo1b at DIV1 by time-lapse spinning confocal microscopy. The first frame of Video 2 and kymographs of 36 frames for the region marked on the first frame by a white lane for the two recombinant proteins are shown. The increase of fluorescence intensity for AKT-PH-EGFP correlated with the increase of fluorescence intensity for mCherry-Myo1b. Bars: (A) 10 µm; (B and C) 20 µm. T, time

    Journal: The Journal of Cell Biology

    Article Title: Myosin 1b promotes axon formation by regulating actin wave propagation and growth cone dynamics

    doi: 10.1083/jcb.201703205

    Figure Lengend Snippet: Myo1b is associated with and propagates with actin waves. (A) Merged fluorescence images acquired with SIM of an actin wave costained with anti-Myo1b antibodies, Alexa Fluor 488 phalloidin, and anti–β3-tubulin antibody and images of the 2.5× enlargement of the region marked by a white box are shown. (B) The propagation of F-actin structures and mCherry-Myo1b has been analyzed in cortical neurons expressing LifeAct-EGFP and mCherry-Myo1b at DIV1 by time-lapse spinning confocal microscopy. The first frame of Video 1 (one frame per minute) for LifeAct-EGFP and mCherry-Myo1b and the merged kymographs of 36 frames for the region marked on the first frame by a white lane for the two recombinant proteins are shown. The increase of fluorescence intensity for LifeAct-EGFP correlated with the increase of fluorescence intensity for mCherry-Myo1b. (C) The propagation of PIP3 and mCherry-Myo1b has been analyzed in cortical neurons expressing AKT-PH-EGFP and mCherry-Myo1b at DIV1 by time-lapse spinning confocal microscopy. The first frame of Video 2 and kymographs of 36 frames for the region marked on the first frame by a white lane for the two recombinant proteins are shown. The increase of fluorescence intensity for AKT-PH-EGFP correlated with the increase of fluorescence intensity for mCherry-Myo1b. Bars: (A) 10 µm; (B and C) 20 µm. T, time

    Article Snippet: Alexa Fluor 488– or 546–conjugated phalloidin was used to detect F-actin (1:400; Invitrogen).

    Techniques: Fluorescence, Expressing, Confocal Microscopy, Recombinant

    Panel A shows confocal images demonstrating membrane binding (arrows) and internalization (arrow heads) of Alexa Fluor 546 (A546) labeled TCRm RL6A by hCMEC/D3 cells (red fluorescence signal). Cells were paraformaldehyde-fixed after 15 min incubation with the antibodies at 37°C. No staining was detectable with an A546 labeled isotype control antibody. In this panel and all other panels nuclei were stained with the far-red DNA dye DRAQ5 and pseudo-colored in blue. DIC = differential interference contrast. Panel B indicates that the TCRm RL6A and the pan HLA-A2 antibody BB7.2 (both A546 labeled, red fluorescence signal) were internalized into endosomal compartments: the arrows and arrowheads in the top group of confocal images highlight colocalization of RL6A or BB7.2, respectively, with the early endosomal marker EEA1 labeled by Alexa Fluor 488 (A488, green fluorescence signal), as evident from the merged images (yellow color). In contrast, the bottom group of images shows lack of colocalization with caveolin 1 (CAV1) by labeling of distinct structures, which do not overlay in the merged images. The hCMEC/D3 cells were incubated with A546 labeled antibodies RL6A or BB7.2 for 30 min at 37°C, followed by fixation and staining for EEA1 or CAV1 (both detected by A488 labeled secondary antibodies). Panel C depicts images of live hCMEC/D3 cells after 30 min of incubation with A546-labeled RL6A, BB7.2 or isotype control antibody. Both RL6A and BB7.2 bind to the cell membrane (arrows) and undergo internalization (arrowheads), while the isotype control showed no interaction with the cells. Panel D compares cellular uptake of RL6A after pre-incubation of the antibody with either an irrelevant peptide/A2 complex in solution (denoted KLM tetramer) or with the cognate YLL/A2 complex (YLL tetramer). Uptake is virtually abolished by YLL tetramer, but unaffected by the irrelevant epitope. The Z-stack provides further evidence for the internalization process of the labeled TCRm (here after 30 min of incubation with the cells). The arrowhead highlights one of the vesicles with clear intracellular localization. Scale bars in all panels = 5 μm.

    Journal: Journal of cellular physiology

    Article Title: A novel vascular targeting strategy for brain-derived endothelial cells using a TCR mimic antibody

    doi: 10.1002/jcp.22256

    Figure Lengend Snippet: Panel A shows confocal images demonstrating membrane binding (arrows) and internalization (arrow heads) of Alexa Fluor 546 (A546) labeled TCRm RL6A by hCMEC/D3 cells (red fluorescence signal). Cells were paraformaldehyde-fixed after 15 min incubation with the antibodies at 37°C. No staining was detectable with an A546 labeled isotype control antibody. In this panel and all other panels nuclei were stained with the far-red DNA dye DRAQ5 and pseudo-colored in blue. DIC = differential interference contrast. Panel B indicates that the TCRm RL6A and the pan HLA-A2 antibody BB7.2 (both A546 labeled, red fluorescence signal) were internalized into endosomal compartments: the arrows and arrowheads in the top group of confocal images highlight colocalization of RL6A or BB7.2, respectively, with the early endosomal marker EEA1 labeled by Alexa Fluor 488 (A488, green fluorescence signal), as evident from the merged images (yellow color). In contrast, the bottom group of images shows lack of colocalization with caveolin 1 (CAV1) by labeling of distinct structures, which do not overlay in the merged images. The hCMEC/D3 cells were incubated with A546 labeled antibodies RL6A or BB7.2 for 30 min at 37°C, followed by fixation and staining for EEA1 or CAV1 (both detected by A488 labeled secondary antibodies). Panel C depicts images of live hCMEC/D3 cells after 30 min of incubation with A546-labeled RL6A, BB7.2 or isotype control antibody. Both RL6A and BB7.2 bind to the cell membrane (arrows) and undergo internalization (arrowheads), while the isotype control showed no interaction with the cells. Panel D compares cellular uptake of RL6A after pre-incubation of the antibody with either an irrelevant peptide/A2 complex in solution (denoted KLM tetramer) or with the cognate YLL/A2 complex (YLL tetramer). Uptake is virtually abolished by YLL tetramer, but unaffected by the irrelevant epitope. The Z-stack provides further evidence for the internalization process of the labeled TCRm (here after 30 min of incubation with the cells). The arrowhead highlights one of the vesicles with clear intracellular localization. Scale bars in all panels = 5 μm.

    Article Snippet: RL-6A, BB7.2 and isotype controls were all labeled to similar specific fluorescence with Alexa Fluor-546 using a commercial kit according to manufacturer's protocol (Invitrogen, Carlsbad, CA) and incubated with 10μg/ml in complete media for different time periods at 37°C.

    Techniques: Binding Assay, Labeling, Fluorescence, Incubation, Staining, Marker

    Generation of antioxidant over-expressing parasites. Transgenic parasites were created in the RHΔhxgprt background strain using plasmids designed to over-express HA-tagged versions of glutaredoxin (Glut), catalase (Cat), or peroxiredoxin 2 (PRx2) under the control of the gra1 gene promoter. ( a ) Over-expression was verified using quantitative real-time PCR to measure transcript levels. ( b ) Western blot analysis was used to confirm protein production and that the resultant proteins were the appropriate size. ( c ) Immunofluorescence assays were performed for protein localization, utilizing a rabbit monoclonal antibody against the HA tag and an Alexa Fluor 594 secondary antibody. ( d ) General parasite replication was determined by doubling assay. Confluent HFF in 24-well plates were infected with the indicated parasite lines for 5 hours before the medium was replaced with fresh growth medium. Parasites were allowed to grow for 32 hours before being methanol-fixed and stained with DiffQuick. The number of individual parasites per vacuole was enumerated for at least 50 vacuoles. Data is presented as percent of vacuoles from 4 experiments. Student’s t-test was employed for statistical analysis. Scale bars equal 2 μm.

    Journal: Scientific Reports

    Article Title: Oxidative stress generated during monensin treatment contributes to altered Toxoplasma gondii mitochondrial function

    doi: 10.1038/srep22997

    Figure Lengend Snippet: Generation of antioxidant over-expressing parasites. Transgenic parasites were created in the RHΔhxgprt background strain using plasmids designed to over-express HA-tagged versions of glutaredoxin (Glut), catalase (Cat), or peroxiredoxin 2 (PRx2) under the control of the gra1 gene promoter. ( a ) Over-expression was verified using quantitative real-time PCR to measure transcript levels. ( b ) Western blot analysis was used to confirm protein production and that the resultant proteins were the appropriate size. ( c ) Immunofluorescence assays were performed for protein localization, utilizing a rabbit monoclonal antibody against the HA tag and an Alexa Fluor 594 secondary antibody. ( d ) General parasite replication was determined by doubling assay. Confluent HFF in 24-well plates were infected with the indicated parasite lines for 5 hours before the medium was replaced with fresh growth medium. Parasites were allowed to grow for 32 hours before being methanol-fixed and stained with DiffQuick. The number of individual parasites per vacuole was enumerated for at least 50 vacuoles. Data is presented as percent of vacuoles from 4 experiments. Student’s t-test was employed for statistical analysis. Scale bars equal 2 μm.

    Article Snippet: Primary antibodies were visualized using either Alexa Fluor 594 or Alexa Fluor 488 secondary antibodies (Life Technologies).

    Techniques: Expressing, Transgenic Assay, Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Infection, Staining

    Characterization of VCAM-1 + macrophages in the CHT. a, Transgenic Tg(kdrl:eGFP) embryos, stained with an anti-VCAM-1 antibody (magenta, arrows), show that the VCAM-1 + macrophage first appeared in the CHT at 32 h.p.f. b, Tg(kdrl:eGFP) embryos in the vcam1 cas011 , itga4 cas005 or runx1 w84x mutant background are stained with an anti-VCAM-1 antibody (magenta, white arrows) at 54 h.p.f. Signals in itga4 cas005 and runx1 w84x are similar to that in wild-type siblings, whereas there is almost no detectable signal in vcam1 cas011 mutants. c, Schematic diagrams (left) and confocal imaging (right) of VCAM-1 + macrophages (labelled with Alexa Fluor 647 dye-conjugated anti-VCAM-1 antibody by intravascular injection) that patrol the CHT of wild-type embryos. VCAM-1 + macrophages were mainly located intravascularly ( > 91%) with round or unpolarized cell morphology ( > 84%). Cross indicates the original position of VCAM-1 + . DA, dorsal aorta; VC, venous capillaries. Scale bars, 50 μm ( a, b ) and 20 μm ( c ).

    Journal: Nature

    Article Title: VCAM-1+ macrophages guide the homing of HSPCs to a vascular niche

    doi: 10.1038/s41586-018-0709-7

    Figure Lengend Snippet: Characterization of VCAM-1 + macrophages in the CHT. a, Transgenic Tg(kdrl:eGFP) embryos, stained with an anti-VCAM-1 antibody (magenta, arrows), show that the VCAM-1 + macrophage first appeared in the CHT at 32 h.p.f. b, Tg(kdrl:eGFP) embryos in the vcam1 cas011 , itga4 cas005 or runx1 w84x mutant background are stained with an anti-VCAM-1 antibody (magenta, white arrows) at 54 h.p.f. Signals in itga4 cas005 and runx1 w84x are similar to that in wild-type siblings, whereas there is almost no detectable signal in vcam1 cas011 mutants. c, Schematic diagrams (left) and confocal imaging (right) of VCAM-1 + macrophages (labelled with Alexa Fluor 647 dye-conjugated anti-VCAM-1 antibody by intravascular injection) that patrol the CHT of wild-type embryos. VCAM-1 + macrophages were mainly located intravascularly ( > 91%) with round or unpolarized cell morphology ( > 84%). Cross indicates the original position of VCAM-1 + . DA, dorsal aorta; VC, venous capillaries. Scale bars, 50 μm ( a, b ) and 20 μm ( c ).

    Article Snippet: For live labelling of usher cells, VCAM-1 antibody (Abclonal) was conjugated with Alexa Fluor 647 dye and purified by Microscale Protein Labelling Kit (Invitrogen, A30009).

    Techniques: Transgenic Assay, Staining, Mutagenesis, Imaging, Injection

    Confocal LSM images of DCs after LPK NP uptake, in which KLH was labeled with Alexa Fluor ® 647, and lipids were labeled with NBD. Dendritic cells were treated with (A) LPK − NPs, (B) LPK + NPs, and (C) LPK pH NPs, respectively. The intracellular

    Journal: Polymer

    Article Title: In vitro controlled release of antigen in dendritic cells using pH-sensitive liposome-polymeric hybrid nanoparticles

    doi: 10.1016/j.polymer.2015.10.048

    Figure Lengend Snippet: Confocal LSM images of DCs after LPK NP uptake, in which KLH was labeled with Alexa Fluor ® 647, and lipids were labeled with NBD. Dendritic cells were treated with (A) LPK − NPs, (B) LPK + NPs, and (C) LPK pH NPs, respectively. The intracellular

    Article Snippet: Fetal bovine serum (FBS), GM-CSF recombinant mouse protein, minimum essential medium (MEM) α , trypsin/EDTA, CellMask™ Orange Plasma membrane Stain, Alexa Fluor® 647 Hydrazide, tris(triethylammonium) salt were purchased from Life Technologies Corporation (Grand Island, NY).

    Techniques: Labeling

    Confocal LSM images of LPK pH NPs, in which KLH was labeled with Alexa Fluor ® 647, and lipids were labeled with NBD.

    Journal: Polymer

    Article Title: In vitro controlled release of antigen in dendritic cells using pH-sensitive liposome-polymeric hybrid nanoparticles

    doi: 10.1016/j.polymer.2015.10.048

    Figure Lengend Snippet: Confocal LSM images of LPK pH NPs, in which KLH was labeled with Alexa Fluor ® 647, and lipids were labeled with NBD.

    Article Snippet: Fetal bovine serum (FBS), GM-CSF recombinant mouse protein, minimum essential medium (MEM) α , trypsin/EDTA, CellMask™ Orange Plasma membrane Stain, Alexa Fluor® 647 Hydrazide, tris(triethylammonium) salt were purchased from Life Technologies Corporation (Grand Island, NY).

    Techniques: Labeling