snap tag alexafluor 647  (New England Biolabs)


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    Name:
    SNAP Surface Alexa Fluor 647
    Description:
    SNAP Surface Alexa Fluor 647 50 nmol
    Catalog Number:
    S9136S
    Price:
    380
    Category:
    Fluorochromes
    Size:
    50 nmol
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    New England Biolabs snap tag alexafluor 647
    SNAP Surface Alexa Fluor 647
    SNAP Surface Alexa Fluor 647 50 nmol
    https://www.bioz.com/result/snap tag alexafluor 647/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    snap tag alexafluor 647 - by Bioz Stars, 2021-07
    99/100 stars

    Images

    1) Product Images from "Complex Formation between VEGFR2 and the β2-Adrenoceptor"

    Article Title: Complex Formation between VEGFR2 and the β2-Adrenoceptor

    Journal: Cell Chemical Biology

    doi: 10.1016/j.chembiol.2019.02.014

    Effect of Agonist Stimulation on Receptor Oligomerization (A) Schematic of experimental setup to investigate the effect of isoprenaline or VEGF 165 a on receptor oligomerization measured using NanoBRET. (B) Visualization of VEGFR2/β 2 -adrenoceptor oligomers by NanoBRET using a luminescence LV200 Olympus microscope. HEK293 cells were transiently co-transfected to express NLuc-tagged-VEGFR2 and SNAP-tagged β 2 -adrenoceptors. Sequential images were captured from unlabeled (top panels) or SNAP-surface AF647-labeled co-transfected cells (bottom panels). Sequential images were acquired by capturing DAPI channel, displayed in the left panels (donor detection; using a 438/24 nm emission filter, 5 s exposure time), followed by CY5 channel, displayed in the right panels (BRET-excited acceptor, using a 647 long-pass filter, 30 s exposure time). Scale bar represents 20 μm. (C and D) HEK293 cells were transiently transfected with 0.05 μg/well NLuc-VEGFR2 and 0.10 μg/well SNAP-β 2 -AR and treated for 1 h at 37°C with increasing concentrations of (C) VEGF 165 a or (D) isoprenaline. Bar C corresponds to untreated (control) condition. Data are means ± SEM from five separate experiments, each performed in quadruplicate. **p
    Figure Legend Snippet: Effect of Agonist Stimulation on Receptor Oligomerization (A) Schematic of experimental setup to investigate the effect of isoprenaline or VEGF 165 a on receptor oligomerization measured using NanoBRET. (B) Visualization of VEGFR2/β 2 -adrenoceptor oligomers by NanoBRET using a luminescence LV200 Olympus microscope. HEK293 cells were transiently co-transfected to express NLuc-tagged-VEGFR2 and SNAP-tagged β 2 -adrenoceptors. Sequential images were captured from unlabeled (top panels) or SNAP-surface AF647-labeled co-transfected cells (bottom panels). Sequential images were acquired by capturing DAPI channel, displayed in the left panels (donor detection; using a 438/24 nm emission filter, 5 s exposure time), followed by CY5 channel, displayed in the right panels (BRET-excited acceptor, using a 647 long-pass filter, 30 s exposure time). Scale bar represents 20 μm. (C and D) HEK293 cells were transiently transfected with 0.05 μg/well NLuc-VEGFR2 and 0.10 μg/well SNAP-β 2 -AR and treated for 1 h at 37°C with increasing concentrations of (C) VEGF 165 a or (D) isoprenaline. Bar C corresponds to untreated (control) condition. Data are means ± SEM from five separate experiments, each performed in quadruplicate. **p

    Techniques Used: Microscopy, Transfection, Labeling, Bioluminescence Resonance Energy Transfer

    Influence of Agonists on the Cellular Location of Receptors and on Complex Formation between β 2 -Adrenoceptors and β-Arrestin2 (A) Confocal imaging (Zeiss LSM 710) of HEK293 cells transiently co-transfected with 0.25 μg/well HaloTag- VEGFR2 and 0.25 μg/well SNAP-β 2 -AR cDNAs, under unstimulated conditions (vehicle) or after treatment with 10 μM isoprenaline or 10 nM VEGF 165 a ligands (30 min at 37°C). Data are representative of three individual experiments. Scale bar represents 20 μm. (B) Immunolabeling of early endosomes (anti-Rab 5 antibody labeling). HEK293 cells transiently co-transfected with 0.5 μg/well HaloTag-VEGFR2 (green) and 0.5 μg/well SNAP-β 2 -AR (red) cDNAs, under unstimulated conditions (vehicle) or after treatment with 10 μM isoprenaline or 10 nM VEGF 165 a (30 min at 37°C). Cells were fixed using 3% paraformaldehyde/PBS, permeabilized using Triton X-100 (0.025% in PBS) and Rab 5 endosomal compartments labeled (cyan). Cells were imaged using a LSM880 confocal microscope (Zeiss). Data are representative of three individual experiments. Scale bar represents 10 μm. (C) Structured illumination microscopy (SIM) super-resolution images of HEK293 cells transiently co-transfected with HaloTag-VEGFR2 (green) and SNAP-β 2 -AR (red; 3 μg total cDNA). Cells were incubated with vehicle, 10 μM isoprenaline or 10 nM VEGF 165 a (30 min at 37°C) before fixation and mounting onto microscope slides. Coverslips were imaged using a Zeiss ELYRA PS.1 microscope. Areas of co-localized HaloTag-VEGFR2 and SNAP-β 2 -AR-labeled receptors are shown in yellow. Scale bar represents 10 μm. (D and E) Summary of Pearson's correlation coefficients (D) obtained following co-localization analysis of SIM images of circular regions of interest (ROI) in HEK293 cells co-expressing HaloTag-VEGFR2 and SNAP-β 2 -AR. ROI were placed on areas of fluorescence either at the plasma membrane or intracellular regions of SIM images of HEK293 cells co-expressing HaloTag-VEGFR2 (green; HaloTag AF488 membrane impermeant label) and SNAP-β 2 -AR (red; SNAP AF647 membrane impermeant label). TetraSpeck microspheres (0.1-μm spectral beads stained with four fluorophores: 365/430 nm [blue], 505/515 nm [green], 560/580 nm [orange], and 660/680 nm [red]) were included in each experiment to allow X/Y/Z channel alignment correction in image processing. The Fiji (ImageJ) analysis program CoLoc2 was applied to these ROI (six ROIs for spectral bead images and 12–15 ROIs for all other conditions) and Pearson's correlation coefficients obtained. Values were averaged across all ROI and are expressed as means ± SEM. A Pearson correlation coefficient value of +1 implies a perfect co-occurrence of both green (HaloTag-VEGFR2) and red (SNAP-β 2 -AR) fluorophores. *p
    Figure Legend Snippet: Influence of Agonists on the Cellular Location of Receptors and on Complex Formation between β 2 -Adrenoceptors and β-Arrestin2 (A) Confocal imaging (Zeiss LSM 710) of HEK293 cells transiently co-transfected with 0.25 μg/well HaloTag- VEGFR2 and 0.25 μg/well SNAP-β 2 -AR cDNAs, under unstimulated conditions (vehicle) or after treatment with 10 μM isoprenaline or 10 nM VEGF 165 a ligands (30 min at 37°C). Data are representative of three individual experiments. Scale bar represents 20 μm. (B) Immunolabeling of early endosomes (anti-Rab 5 antibody labeling). HEK293 cells transiently co-transfected with 0.5 μg/well HaloTag-VEGFR2 (green) and 0.5 μg/well SNAP-β 2 -AR (red) cDNAs, under unstimulated conditions (vehicle) or after treatment with 10 μM isoprenaline or 10 nM VEGF 165 a (30 min at 37°C). Cells were fixed using 3% paraformaldehyde/PBS, permeabilized using Triton X-100 (0.025% in PBS) and Rab 5 endosomal compartments labeled (cyan). Cells were imaged using a LSM880 confocal microscope (Zeiss). Data are representative of three individual experiments. Scale bar represents 10 μm. (C) Structured illumination microscopy (SIM) super-resolution images of HEK293 cells transiently co-transfected with HaloTag-VEGFR2 (green) and SNAP-β 2 -AR (red; 3 μg total cDNA). Cells were incubated with vehicle, 10 μM isoprenaline or 10 nM VEGF 165 a (30 min at 37°C) before fixation and mounting onto microscope slides. Coverslips were imaged using a Zeiss ELYRA PS.1 microscope. Areas of co-localized HaloTag-VEGFR2 and SNAP-β 2 -AR-labeled receptors are shown in yellow. Scale bar represents 10 μm. (D and E) Summary of Pearson's correlation coefficients (D) obtained following co-localization analysis of SIM images of circular regions of interest (ROI) in HEK293 cells co-expressing HaloTag-VEGFR2 and SNAP-β 2 -AR. ROI were placed on areas of fluorescence either at the plasma membrane or intracellular regions of SIM images of HEK293 cells co-expressing HaloTag-VEGFR2 (green; HaloTag AF488 membrane impermeant label) and SNAP-β 2 -AR (red; SNAP AF647 membrane impermeant label). TetraSpeck microspheres (0.1-μm spectral beads stained with four fluorophores: 365/430 nm [blue], 505/515 nm [green], 560/580 nm [orange], and 660/680 nm [red]) were included in each experiment to allow X/Y/Z channel alignment correction in image processing. The Fiji (ImageJ) analysis program CoLoc2 was applied to these ROI (six ROIs for spectral bead images and 12–15 ROIs for all other conditions) and Pearson's correlation coefficients obtained. Values were averaged across all ROI and are expressed as means ± SEM. A Pearson correlation coefficient value of +1 implies a perfect co-occurrence of both green (HaloTag-VEGFR2) and red (SNAP-β 2 -AR) fluorophores. *p

    Techniques Used: Imaging, Transfection, Immunolabeling, Antibody Labeling, Labeling, Microscopy, Incubation, Expressing, Fluorescence, Staining

    Related Articles

    Purification:

    Article Title: Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen
    Article Snippet: The protein concentration was determined using the AIDA Image Analyzer as described above. .. Coupling SNAP-TTC to the fluorescent dye Purified SNAP-TTC was conjugated to the BG-modified fluorescent dyes SNAP-surface® Alexa Fluor® 647 (New England Biolabs, Frankfurt am Main, Germany; Catalogue number: S9136S) and SNAP-surface® Alexa Fluor® 488 (New England Biolabs; Catalogue number: S9129S) as previously described [ ]. .. Briefly, 1 μg SNAP-TTC protein was mixed with 2 nmol BG-647 or BG-488 solution prepared from a 50 nmol stock and incubated for 1 h at room temperature.

    Staining:

    Article Title: Antigen-Induced Allosteric Changes in a Human IgG1 Fc Increase Low-Affinity Fcγ Receptor Binding
    Article Snippet: ADCC was assessed using the rapid fluorescence antibody-dependent cellular cytotoxicity assay (RFADCC) ( ) as modified in our laboratory for high-throughput assays ( ). .. Briefly, EGFP-CEM-NKr-CCR5SNAP cells stained with SNAP-Surface Alexa Fluor 647 (New England BioLabs) and sensitized with recombinant HIV-1 Ba-L gp120 were used as targets and PBMC were utilized as effectors. .. All the antibodies activity was analyzed with four-fold serial dilutions starting from a concentration of 0.3 ug/ml.

    Article Title: Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis
    Article Snippet: At 24-hour post seeding, the cells were transfected with 100 ng of either SNAP-FL, SNAP-ΔG, or SNAP-ΔGΔH-EAP45 expressor. .. This was followed by fixation with PFA and staining with SNAP-Surface AlexaFluor647 (NEB) at 4 µM concentration for SNAP-EAP45 and DAPI for nuclei on the following day. .. A primary antibody to RAB7 (Abcam) or anti-Tubulin (Sigma) was also added on some occasions followed by staining with the secondary antibody conjugated with either AlexaFluor488 or AlexaFluor647 (Thermofisher) before imaging using Leica SP5 confocal fluorescence microscope.

    Article Title: Correlative Light- and Electron Microscopy with chemical tags
    Article Snippet: L cells stably expressing Ncad-SNAPf were grown to a confluency of 60–80%, stained in 2 μM SNAP-Surface Alexa Fluor 647 solution (dilution in phenolred free complete medium) at 37 °C for 5 min and subsequently washed three times with phenolred free complete medium. .. For staining of HeLa cells, 3 μM SNAP-Cell TMR Star, 2 μM SNAP-Surface Alexa Fluor 647 (New England BioLabs), 3 μM SiR-SNAP ( ) and 50 μM HaloTag TMR Ligand (Promega) diluted in OptiMEM (Life Technologies) with 10% FBS were used. .. After 30 min of staining, samples were rinsed three times and washed for at least 30 min with OptiMEM with 10% FBS.

    Article Title: A new cell line for high throughput HIV-specific antibody-dependent cellular cytotoxicity (ADCC) and cell-to-cell virus transmission studies
    Article Snippet: 2.3 Optimized RFADCC assay and cell surface staining To optimize the RFADCC assay ( ) for high throughput efficiency, the regular double staining with the membrane PKH-26 dye and the intracellular carboxyfluorescein diacetate, succinimidyl ester (CSFE) dye were replaced with the membrane staining of the CCR5-SNAP-tag and the constitutive intracellular expression of GFP. .. For the ADCC protocol , EGFP-CEM-NKr-CCR5-SNAP target cells were stained with the fluorescent substrate SNAP-Surface Alexa Fluor 647 (New England BioLabs Cat. S9136S) for 20 min at 37 °C with or without coating of monomeric HIV-1 Bal gp120 (50 μg/ml). .. For the studies with spinoculated virus, the cells were first stained with the SNAP-Surface dye and then spinoculated with the AT-2 inactivated Bal HIV-1 virus at 2000 RPM for 2 h at 12 °C.

    Recombinant:

    Article Title: Antigen-Induced Allosteric Changes in a Human IgG1 Fc Increase Low-Affinity Fcγ Receptor Binding
    Article Snippet: ADCC was assessed using the rapid fluorescence antibody-dependent cellular cytotoxicity assay (RFADCC) ( ) as modified in our laboratory for high-throughput assays ( ). .. Briefly, EGFP-CEM-NKr-CCR5SNAP cells stained with SNAP-Surface Alexa Fluor 647 (New England BioLabs) and sensitized with recombinant HIV-1 Ba-L gp120 were used as targets and PBMC were utilized as effectors. .. All the antibodies activity was analyzed with four-fold serial dilutions starting from a concentration of 0.3 ug/ml.

    Concentration Assay:

    Article Title: Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis
    Article Snippet: At 24-hour post seeding, the cells were transfected with 100 ng of either SNAP-FL, SNAP-ΔG, or SNAP-ΔGΔH-EAP45 expressor. .. This was followed by fixation with PFA and staining with SNAP-Surface AlexaFluor647 (NEB) at 4 µM concentration for SNAP-EAP45 and DAPI for nuclei on the following day. .. A primary antibody to RAB7 (Abcam) or anti-Tubulin (Sigma) was also added on some occasions followed by staining with the secondary antibody conjugated with either AlexaFluor488 or AlexaFluor647 (Thermofisher) before imaging using Leica SP5 confocal fluorescence microscope.

    Incubation:

    Article Title: Exclusive formation of monovalent quantum dot imaging probes by steric exclusion
    Article Snippet: Live cell labeling and imaging Unless otherwise noted, cells were incubated with 1 μM BG-DNA for 30 minutes at 37 °C, washed three times with PBS containing 1 % BSA, and then incubated with 200 pM mQDs for 5 minutes at room temperature before a final wash with 1 % BSA. .. In dye-comparison experiments, cells were incubated simultaneously with 1 μM BG-DNA and 0.2 μM BG-AF647 (Surface-SNAP-647, NEB). .. For single particle tracking, mQDs were incubated in PBS containing 1 % BSA for 30 min at room temperature prior to being added to cells at a concentration of 0.2 nM.

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    New England Biolabs snap tag alexafluor 647
    Effect of Agonist Stimulation on Receptor Oligomerization (A) Schematic of experimental setup to investigate the effect of isoprenaline or VEGF 165 a on receptor oligomerization measured using NanoBRET. (B) Visualization of VEGFR2/β 2 -adrenoceptor oligomers by NanoBRET using a luminescence LV200 Olympus microscope. HEK293 cells were transiently co-transfected to express NLuc-tagged-VEGFR2 and SNAP-tagged β 2 -adrenoceptors. Sequential images were captured from unlabeled (top panels) or SNAP-surface <t>AF647-labeled</t> co-transfected cells (bottom panels). Sequential images were acquired by capturing DAPI channel, displayed in the left panels (donor detection; using a 438/24 nm emission filter, 5 s exposure time), followed by CY5 channel, displayed in the right panels (BRET-excited acceptor, using a 647 long-pass filter, 30 s exposure time). Scale bar represents 20 μm. (C and D) HEK293 cells were transiently transfected with 0.05 μg/well NLuc-VEGFR2 and 0.10 μg/well SNAP-β 2 -AR and treated for 1 h at 37°C with increasing concentrations of (C) VEGF 165 a or (D) isoprenaline. Bar C corresponds to untreated (control) condition. Data are means ± SEM from five separate experiments, each performed in quadruplicate. **p
    Snap Tag Alexafluor 647, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/snap tag alexafluor 647/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    snap tag alexafluor 647 - by Bioz Stars, 2021-07
    99/100 stars
      Buy from Supplier

    Image Search Results


    Effect of Agonist Stimulation on Receptor Oligomerization (A) Schematic of experimental setup to investigate the effect of isoprenaline or VEGF 165 a on receptor oligomerization measured using NanoBRET. (B) Visualization of VEGFR2/β 2 -adrenoceptor oligomers by NanoBRET using a luminescence LV200 Olympus microscope. HEK293 cells were transiently co-transfected to express NLuc-tagged-VEGFR2 and SNAP-tagged β 2 -adrenoceptors. Sequential images were captured from unlabeled (top panels) or SNAP-surface AF647-labeled co-transfected cells (bottom panels). Sequential images were acquired by capturing DAPI channel, displayed in the left panels (donor detection; using a 438/24 nm emission filter, 5 s exposure time), followed by CY5 channel, displayed in the right panels (BRET-excited acceptor, using a 647 long-pass filter, 30 s exposure time). Scale bar represents 20 μm. (C and D) HEK293 cells were transiently transfected with 0.05 μg/well NLuc-VEGFR2 and 0.10 μg/well SNAP-β 2 -AR and treated for 1 h at 37°C with increasing concentrations of (C) VEGF 165 a or (D) isoprenaline. Bar C corresponds to untreated (control) condition. Data are means ± SEM from five separate experiments, each performed in quadruplicate. **p

    Journal: Cell Chemical Biology

    Article Title: Complex Formation between VEGFR2 and the β2-Adrenoceptor

    doi: 10.1016/j.chembiol.2019.02.014

    Figure Lengend Snippet: Effect of Agonist Stimulation on Receptor Oligomerization (A) Schematic of experimental setup to investigate the effect of isoprenaline or VEGF 165 a on receptor oligomerization measured using NanoBRET. (B) Visualization of VEGFR2/β 2 -adrenoceptor oligomers by NanoBRET using a luminescence LV200 Olympus microscope. HEK293 cells were transiently co-transfected to express NLuc-tagged-VEGFR2 and SNAP-tagged β 2 -adrenoceptors. Sequential images were captured from unlabeled (top panels) or SNAP-surface AF647-labeled co-transfected cells (bottom panels). Sequential images were acquired by capturing DAPI channel, displayed in the left panels (donor detection; using a 438/24 nm emission filter, 5 s exposure time), followed by CY5 channel, displayed in the right panels (BRET-excited acceptor, using a 647 long-pass filter, 30 s exposure time). Scale bar represents 20 μm. (C and D) HEK293 cells were transiently transfected with 0.05 μg/well NLuc-VEGFR2 and 0.10 μg/well SNAP-β 2 -AR and treated for 1 h at 37°C with increasing concentrations of (C) VEGF 165 a or (D) isoprenaline. Bar C corresponds to untreated (control) condition. Data are means ± SEM from five separate experiments, each performed in quadruplicate. **p

    Article Snippet: SNAP-Tag® AlexaFluor 647 (AF647) and AlexaFluor 488 (AF488) were purchased from New England BioLabs (Massachusetts, USA).

    Techniques: Microscopy, Transfection, Labeling, Bioluminescence Resonance Energy Transfer

    Influence of Agonists on the Cellular Location of Receptors and on Complex Formation between β 2 -Adrenoceptors and β-Arrestin2 (A) Confocal imaging (Zeiss LSM 710) of HEK293 cells transiently co-transfected with 0.25 μg/well HaloTag- VEGFR2 and 0.25 μg/well SNAP-β 2 -AR cDNAs, under unstimulated conditions (vehicle) or after treatment with 10 μM isoprenaline or 10 nM VEGF 165 a ligands (30 min at 37°C). Data are representative of three individual experiments. Scale bar represents 20 μm. (B) Immunolabeling of early endosomes (anti-Rab 5 antibody labeling). HEK293 cells transiently co-transfected with 0.5 μg/well HaloTag-VEGFR2 (green) and 0.5 μg/well SNAP-β 2 -AR (red) cDNAs, under unstimulated conditions (vehicle) or after treatment with 10 μM isoprenaline or 10 nM VEGF 165 a (30 min at 37°C). Cells were fixed using 3% paraformaldehyde/PBS, permeabilized using Triton X-100 (0.025% in PBS) and Rab 5 endosomal compartments labeled (cyan). Cells were imaged using a LSM880 confocal microscope (Zeiss). Data are representative of three individual experiments. Scale bar represents 10 μm. (C) Structured illumination microscopy (SIM) super-resolution images of HEK293 cells transiently co-transfected with HaloTag-VEGFR2 (green) and SNAP-β 2 -AR (red; 3 μg total cDNA). Cells were incubated with vehicle, 10 μM isoprenaline or 10 nM VEGF 165 a (30 min at 37°C) before fixation and mounting onto microscope slides. Coverslips were imaged using a Zeiss ELYRA PS.1 microscope. Areas of co-localized HaloTag-VEGFR2 and SNAP-β 2 -AR-labeled receptors are shown in yellow. Scale bar represents 10 μm. (D and E) Summary of Pearson's correlation coefficients (D) obtained following co-localization analysis of SIM images of circular regions of interest (ROI) in HEK293 cells co-expressing HaloTag-VEGFR2 and SNAP-β 2 -AR. ROI were placed on areas of fluorescence either at the plasma membrane or intracellular regions of SIM images of HEK293 cells co-expressing HaloTag-VEGFR2 (green; HaloTag AF488 membrane impermeant label) and SNAP-β 2 -AR (red; SNAP AF647 membrane impermeant label). TetraSpeck microspheres (0.1-μm spectral beads stained with four fluorophores: 365/430 nm [blue], 505/515 nm [green], 560/580 nm [orange], and 660/680 nm [red]) were included in each experiment to allow X/Y/Z channel alignment correction in image processing. The Fiji (ImageJ) analysis program CoLoc2 was applied to these ROI (six ROIs for spectral bead images and 12–15 ROIs for all other conditions) and Pearson's correlation coefficients obtained. Values were averaged across all ROI and are expressed as means ± SEM. A Pearson correlation coefficient value of +1 implies a perfect co-occurrence of both green (HaloTag-VEGFR2) and red (SNAP-β 2 -AR) fluorophores. *p

    Journal: Cell Chemical Biology

    Article Title: Complex Formation between VEGFR2 and the β2-Adrenoceptor

    doi: 10.1016/j.chembiol.2019.02.014

    Figure Lengend Snippet: Influence of Agonists on the Cellular Location of Receptors and on Complex Formation between β 2 -Adrenoceptors and β-Arrestin2 (A) Confocal imaging (Zeiss LSM 710) of HEK293 cells transiently co-transfected with 0.25 μg/well HaloTag- VEGFR2 and 0.25 μg/well SNAP-β 2 -AR cDNAs, under unstimulated conditions (vehicle) or after treatment with 10 μM isoprenaline or 10 nM VEGF 165 a ligands (30 min at 37°C). Data are representative of three individual experiments. Scale bar represents 20 μm. (B) Immunolabeling of early endosomes (anti-Rab 5 antibody labeling). HEK293 cells transiently co-transfected with 0.5 μg/well HaloTag-VEGFR2 (green) and 0.5 μg/well SNAP-β 2 -AR (red) cDNAs, under unstimulated conditions (vehicle) or after treatment with 10 μM isoprenaline or 10 nM VEGF 165 a (30 min at 37°C). Cells were fixed using 3% paraformaldehyde/PBS, permeabilized using Triton X-100 (0.025% in PBS) and Rab 5 endosomal compartments labeled (cyan). Cells were imaged using a LSM880 confocal microscope (Zeiss). Data are representative of three individual experiments. Scale bar represents 10 μm. (C) Structured illumination microscopy (SIM) super-resolution images of HEK293 cells transiently co-transfected with HaloTag-VEGFR2 (green) and SNAP-β 2 -AR (red; 3 μg total cDNA). Cells were incubated with vehicle, 10 μM isoprenaline or 10 nM VEGF 165 a (30 min at 37°C) before fixation and mounting onto microscope slides. Coverslips were imaged using a Zeiss ELYRA PS.1 microscope. Areas of co-localized HaloTag-VEGFR2 and SNAP-β 2 -AR-labeled receptors are shown in yellow. Scale bar represents 10 μm. (D and E) Summary of Pearson's correlation coefficients (D) obtained following co-localization analysis of SIM images of circular regions of interest (ROI) in HEK293 cells co-expressing HaloTag-VEGFR2 and SNAP-β 2 -AR. ROI were placed on areas of fluorescence either at the plasma membrane or intracellular regions of SIM images of HEK293 cells co-expressing HaloTag-VEGFR2 (green; HaloTag AF488 membrane impermeant label) and SNAP-β 2 -AR (red; SNAP AF647 membrane impermeant label). TetraSpeck microspheres (0.1-μm spectral beads stained with four fluorophores: 365/430 nm [blue], 505/515 nm [green], 560/580 nm [orange], and 660/680 nm [red]) were included in each experiment to allow X/Y/Z channel alignment correction in image processing. The Fiji (ImageJ) analysis program CoLoc2 was applied to these ROI (six ROIs for spectral bead images and 12–15 ROIs for all other conditions) and Pearson's correlation coefficients obtained. Values were averaged across all ROI and are expressed as means ± SEM. A Pearson correlation coefficient value of +1 implies a perfect co-occurrence of both green (HaloTag-VEGFR2) and red (SNAP-β 2 -AR) fluorophores. *p

    Article Snippet: SNAP-Tag® AlexaFluor 647 (AF647) and AlexaFluor 488 (AF488) were purchased from New England BioLabs (Massachusetts, USA).

    Techniques: Imaging, Transfection, Immunolabeling, Antibody Labeling, Labeling, Microscopy, Incubation, Expressing, Fluorescence, Staining