Structured Review

Affibody t47d cells
Fluorescence intensity measured from confocal microscopy images of <t>T47D</t> cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.
T47d Cells, supplied by Affibody, used in various techniques. Bioz Stars score: 92/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/t47d cells/product/Affibody
Average 92 stars, based on 71 article reviews
Price from $9.99 to $1999.99
t47d cells - by Bioz Stars, 2020-09
92/100 stars

Images

1) Product Images from "Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding"

Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074200

Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.
Figure Legend Snippet: Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.

Techniques Used: Fluorescence, Confocal Microscopy, Labeling, Derivative Assay

2) Product Images from "Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding"

Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074200

Mean instantaneous D fit for different anti-EGFR Affibody conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.
Figure Legend Snippet: Mean instantaneous D fit for different anti-EGFR Affibody conjugates. Each datapoint corresponds to mean ± SEM of at least 10 areas acquired from 3 independent samples.

Techniques Used:

Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.
Figure Legend Snippet: Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.

Techniques Used: Fluorescence, Confocal Microscopy, Labeling, Derivative Assay

Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.
Figure Legend Snippet: Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

Techniques Used:

Analysis of definitely mobile vs immobile or very slow moving spots. A) Mean instantaneous D fit for different anti-EGFR Affibody conjugates, after removing data for spots with D values below 0.1 µm 2 /s. Each datapoint corresponds to mean ± SD of of the tracks contained in at least 10 different areas containing a minimum of 50 different cells. Blue bars indicate dyes excited at 491 nm, green at 561 nm, and red at 638 nm. B) Percentages of spots for each dye with D values below 0.1 µm 2 /s. C) Plot of mean instantaneous D fit for different anti-EGFR Affibody conjugates (calculated from all spots) vs percentage of spots with D values
Figure Legend Snippet: Analysis of definitely mobile vs immobile or very slow moving spots. A) Mean instantaneous D fit for different anti-EGFR Affibody conjugates, after removing data for spots with D values below 0.1 µm 2 /s. Each datapoint corresponds to mean ± SD of of the tracks contained in at least 10 different areas containing a minimum of 50 different cells. Blue bars indicate dyes excited at 491 nm, green at 561 nm, and red at 638 nm. B) Percentages of spots for each dye with D values below 0.1 µm 2 /s. C) Plot of mean instantaneous D fit for different anti-EGFR Affibody conjugates (calculated from all spots) vs percentage of spots with D values

Techniques Used:

3) Product Images from "Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding"

Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074200

Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.
Figure Legend Snippet: Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

Techniques Used:

4) Product Images from "A method for comparing intra-tumoural radioactivity uptake heterogeneity in preclinical positron emission tomography studies"

Article Title: A method for comparing intra-tumoural radioactivity uptake heterogeneity in preclinical positron emission tomography studies

Journal: EJNMMI Physics

doi: 10.1186/s40658-015-0124-1

PET transaxial images ( a , b , the colour scales are the same), histograms ( c , d ) of the heterogeneity contributions (the mean intensity deviation per distance calculated according to Eq. 2 ) and surface plots ( e , f ) of the uptake of AnxA5 and mTrx-GFP in a FaDu xenograft. The imaging was performed in the same animal > 2 h apart on the same day. In e and f , the X - and Y -axes represent spatial dimensions and the Z -axis is the mean tracer uptake (SUV mean )
Figure Legend Snippet: PET transaxial images ( a , b , the colour scales are the same), histograms ( c , d ) of the heterogeneity contributions (the mean intensity deviation per distance calculated according to Eq. 2 ) and surface plots ( e , f ) of the uptake of AnxA5 and mTrx-GFP in a FaDu xenograft. The imaging was performed in the same animal > 2 h apart on the same day. In e and f , the X - and Y -axes represent spatial dimensions and the Z -axis is the mean tracer uptake (SUV mean )

Techniques Used: Positron Emission Tomography, Imaging

5) Product Images from "Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents"

Article Title: Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents

Journal: Nature Communications

doi: 10.1038/ncomms15623

Analysis of angiopep-2 transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.
Figure Legend Snippet: Analysis of angiopep-2 transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.

Techniques Used: Fluorescence, Expressing, Staining, Incubation, Concentration Assay, Confocal Microscopy, Permeability, Injection, Mouse Assay

Analysis of angiopep-2 transport using the well-established in vitro BBB Transwell system. Permeability assay using the BBB co-culture Transwell model showing that the ( a ) scrambled control and ( b ) angiopep-2 displayed significantly lower permeation in the co-culture model compared to inserts containing no cells (which represent passive diffusion). ( c ) The Transwell co-culture model failed to differentiate between the permeability of angiopep-2 and the scrambled peptide. For all permeability assays, TAMRA-labelled angiopep-2 (or scramble) peptide (10 μM concentration) was added onto the apical side of the Transwells of the co-culture model after 84 h of incubation. The basal side of the Transwell was imaged using fluorescence microscopy, and the fluorescence intensity was quantified over 40 h. The plots show the accumulation of fluorescence intensity over time with s.d. error bars ( n transwell =2, n experiment =2). Statistical analysis was performed using the one-way ANOVA and Tukey's multiple comparison test. ( d ) Confocal images showing higher expression of ZO-1 (tight junction), P-gp (efflux pump) and β-catenin (adherens junction; shown in white) on the surface of BBB spheroids compared with hCMEC/D3 ECs in the triple co-culture Transwell model after 48 h. Cell nuclei were labelled with Hoechst dye (shown in blue). Scale bar, 100 μm.
Figure Legend Snippet: Analysis of angiopep-2 transport using the well-established in vitro BBB Transwell system. Permeability assay using the BBB co-culture Transwell model showing that the ( a ) scrambled control and ( b ) angiopep-2 displayed significantly lower permeation in the co-culture model compared to inserts containing no cells (which represent passive diffusion). ( c ) The Transwell co-culture model failed to differentiate between the permeability of angiopep-2 and the scrambled peptide. For all permeability assays, TAMRA-labelled angiopep-2 (or scramble) peptide (10 μM concentration) was added onto the apical side of the Transwells of the co-culture model after 84 h of incubation. The basal side of the Transwell was imaged using fluorescence microscopy, and the fluorescence intensity was quantified over 40 h. The plots show the accumulation of fluorescence intensity over time with s.d. error bars ( n transwell =2, n experiment =2). Statistical analysis was performed using the one-way ANOVA and Tukey's multiple comparison test. ( d ) Confocal images showing higher expression of ZO-1 (tight junction), P-gp (efflux pump) and β-catenin (adherens junction; shown in white) on the surface of BBB spheroids compared with hCMEC/D3 ECs in the triple co-culture Transwell model after 48 h. Cell nuclei were labelled with Hoechst dye (shown in blue). Scale bar, 100 μm.

Techniques Used: In Vitro, Permeability, Co-Culture Assay, Diffusion-based Assay, Concentration Assay, Incubation, Fluorescence, Microscopy, Expressing

6) Product Images from "Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin"

Article Title: Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00521

Correlative FM and STEM of whole breast cancer cells. (A,C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B,D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000 ×, D: M = 4,000 ×. (E,F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light gray shade compared to flat cell regions in F, appearing in a darker gray, E: M = 100,000×, F: M = 120,000×.
Figure Legend Snippet: Correlative FM and STEM of whole breast cancer cells. (A,C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B,D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000 ×, D: M = 4,000 ×. (E,F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light gray shade compared to flat cell regions in F, appearing in a darker gray, E: M = 100,000×, F: M = 120,000×.

Techniques Used: Fluorescence, Labeling

Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. (B) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. (D) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).
Figure Legend Snippet: Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. (B) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. (D) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).

Techniques Used: Transduction, Construct, Labeling, Evaporation, Microscopy, Fluorescence, Transmission Assay, Electron Microscopy

Graphs of the pair correlation function g ( r ) versus the radial distance r between QD-labels, collected at actin-rich- and actin-low cell regions with correlating schemes. (A) g ( r ) of QD-labels detected in actin-rich cellular regions collected from 27 images of 11 cells. The red dotted line marks g ( r ) = 20 nm. As comparison, g ( r ) of simulated data is included of randomly positioned labels with the same particle density (see Table 1 ). (B) g ( r ) of QD-labels in actin-low regions collected in 17 images of 8 cells. The arrow marks a peak at r = 36 nm. The red dotted line is at r = 20 nm. g ( r ) of simulated data of randomly positioned labels is also included. (C) Schematic representation of the assumed distribution of ErbB2 and EGFR homo- and heterodimers in relation to cortical actin filaments and lipid rafts in actin-rich, ruffled cell regions. EGFR is bound to actin filaments while ErbB2 can move freely in the membrane and lipid rafts. Those ErbB2 molecules assembled in ErbB2:EGFR heterodimers are bound to helical actin filaments with a pitch of 36 nm. The number of ErbB2 homodimers in ruffled regions outweighs the number of postulated heterodimers. (D) Actin-low, flat cell regions with fewer ErbB2 homodimers being present than in ruffled regions, and postulated heterodimers dominating the analysis. The ruffled regions contain a higher number of growth factor receptors than the flat regions.
Figure Legend Snippet: Graphs of the pair correlation function g ( r ) versus the radial distance r between QD-labels, collected at actin-rich- and actin-low cell regions with correlating schemes. (A) g ( r ) of QD-labels detected in actin-rich cellular regions collected from 27 images of 11 cells. The red dotted line marks g ( r ) = 20 nm. As comparison, g ( r ) of simulated data is included of randomly positioned labels with the same particle density (see Table 1 ). (B) g ( r ) of QD-labels in actin-low regions collected in 17 images of 8 cells. The arrow marks a peak at r = 36 nm. The red dotted line is at r = 20 nm. g ( r ) of simulated data of randomly positioned labels is also included. (C) Schematic representation of the assumed distribution of ErbB2 and EGFR homo- and heterodimers in relation to cortical actin filaments and lipid rafts in actin-rich, ruffled cell regions. EGFR is bound to actin filaments while ErbB2 can move freely in the membrane and lipid rafts. Those ErbB2 molecules assembled in ErbB2:EGFR heterodimers are bound to helical actin filaments with a pitch of 36 nm. The number of ErbB2 homodimers in ruffled regions outweighs the number of postulated heterodimers. (D) Actin-low, flat cell regions with fewer ErbB2 homodimers being present than in ruffled regions, and postulated heterodimers dominating the analysis. The ruffled regions contain a higher number of growth factor receptors than the flat regions.

Techniques Used:

Correlative FM and STEM of whole breast cancer cells treated with Cytochalasin D. (A) Cropped fluorescence micrograph of Cytochalasin D treated SKBR3 cells with actin-GFP in green, QD-labeled membrane ErbB2 in red and overlapping areas in yellow. (B,C) Corresponding STEM images of graphene-covered SKBR3 breast cancer cells ( M = B: 1000×, C: 30,000×). (D) Cytochalasin D inducedindentations are marked exemplarily with white arrowheads. STEM image of region enclosed with dashed square in A ( M = 100,000×). Automatically detected QD-labels are marked in yellow to enhance visibility. (E) g ( r ) of QD-labels collected in seven images of cell edges of Cytochalasin D treated cells. The arrow marks a peak at r = 36 nm, and the dotted line indicates r = 20 nm. g ( r ) of simulated data of randomly positioned labels.
Figure Legend Snippet: Correlative FM and STEM of whole breast cancer cells treated with Cytochalasin D. (A) Cropped fluorescence micrograph of Cytochalasin D treated SKBR3 cells with actin-GFP in green, QD-labeled membrane ErbB2 in red and overlapping areas in yellow. (B,C) Corresponding STEM images of graphene-covered SKBR3 breast cancer cells ( M = B: 1000×, C: 30,000×). (D) Cytochalasin D inducedindentations are marked exemplarily with white arrowheads. STEM image of region enclosed with dashed square in A ( M = 100,000×). Automatically detected QD-labels are marked in yellow to enhance visibility. (E) g ( r ) of QD-labels collected in seven images of cell edges of Cytochalasin D treated cells. The arrow marks a peak at r = 36 nm, and the dotted line indicates r = 20 nm. g ( r ) of simulated data of randomly positioned labels.

Techniques Used: Fluorescence, Labeling

Correlative FM and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M = 30,000×. (D) Region selected from C imaged at M = 100,000×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180 ± 30°.
Figure Legend Snippet: Correlative FM and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M = 30,000×. (D) Region selected from C imaged at M = 100,000×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180 ± 30°.

Techniques Used: Labeling, Fluorescence

7) Product Images from "Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding"

Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074200

Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.
Figure Legend Snippet: Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

Techniques Used:

8) Product Images from "Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin"

Article Title: Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

Journal: bioRxiv

doi: 10.1101/2020.01.14.906040

Correlative fluorescence microscopy and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M =30,000×. (D) Region selected from C imaged at M =100,000 ×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180° ± 30°.
Figure Legend Snippet: Correlative fluorescence microscopy and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M =30,000×. (D) Region selected from C imaged at M =100,000 ×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180° ± 30°.

Techniques Used: Fluorescence, Microscopy, Labeling

Graphs of the pair correlation function g ( r ) versus the radial distance r between QD-labels, collected at actin-rich- and actin-low cell regions with correlating schemes. (A) g ( r ) of QD-labels detected in actin-rich cellular regions collected from 27 images of 11 cells. The red dotted line marks g ( r ) = 20 nm. As comparison, g ( r ) of simulated data is included of randomly positioned labels with the same particle density (see Table 1 ). (B) g ( r ) of QD-labels in actin-low regions collected in 17 images of 8 cells. The arrow marks a peak at r = 36 nm. The red dotted line is at r = 20 nm. g ( r ) of simulated data of randomly positioned labels is also included. (C) Schematic representation of the assumed distribution of ErbB2 and EGFR homo- and heterodimers in relation to cortical actin filaments and lipid rafts in actin-rich, ruffled cell regions. EGFR is bound to actin filaments while ErbB2 can move freely in the membrane and lipid rafts. Those ErbB2 molecules assembled in ErbB2:EGFR heterodimers are bound to helical actin filaments with a pitch of 36 nm. The number of ErbB2 homodimers in ruffled regions outweighs the number of postulated heterodimers. ( D ) Actin-low, flat cell regions with fewer ErbB2 homodimers being present than in ruffled regions, and postulated heterodimers dominating the analysis. The ruffled regions contain a higher number of growth factor receptors than the flat regions.
Figure Legend Snippet: Graphs of the pair correlation function g ( r ) versus the radial distance r between QD-labels, collected at actin-rich- and actin-low cell regions with correlating schemes. (A) g ( r ) of QD-labels detected in actin-rich cellular regions collected from 27 images of 11 cells. The red dotted line marks g ( r ) = 20 nm. As comparison, g ( r ) of simulated data is included of randomly positioned labels with the same particle density (see Table 1 ). (B) g ( r ) of QD-labels in actin-low regions collected in 17 images of 8 cells. The arrow marks a peak at r = 36 nm. The red dotted line is at r = 20 nm. g ( r ) of simulated data of randomly positioned labels is also included. (C) Schematic representation of the assumed distribution of ErbB2 and EGFR homo- and heterodimers in relation to cortical actin filaments and lipid rafts in actin-rich, ruffled cell regions. EGFR is bound to actin filaments while ErbB2 can move freely in the membrane and lipid rafts. Those ErbB2 molecules assembled in ErbB2:EGFR heterodimers are bound to helical actin filaments with a pitch of 36 nm. The number of ErbB2 homodimers in ruffled regions outweighs the number of postulated heterodimers. ( D ) Actin-low, flat cell regions with fewer ErbB2 homodimers being present than in ruffled regions, and postulated heterodimers dominating the analysis. The ruffled regions contain a higher number of growth factor receptors than the flat regions.

Techniques Used:

Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. ( B ) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. ( D ) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).
Figure Legend Snippet: Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. ( B ) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. ( D ) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).

Techniques Used: Transduction, Construct, Labeling, Evaporation, Microscopy, Fluorescence, Transmission Assay, Electron Microscopy

Correlative fluorescence microscopy and STEM of whole breast cancer cells. (A, C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B, D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000×, D: M = 4,000×. (E, F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light grey shade compared to flat cell regions in F, appearing in a darker grey, E: M = 100,000×, F: M = 120,000×.
Figure Legend Snippet: Correlative fluorescence microscopy and STEM of whole breast cancer cells. (A, C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B, D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000×, D: M = 4,000×. (E, F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light grey shade compared to flat cell regions in F, appearing in a darker grey, E: M = 100,000×, F: M = 120,000×.

Techniques Used: Fluorescence, Microscopy, Labeling

9) Product Images from "Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin"

Article Title: Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00521

Correlative FM and STEM of whole breast cancer cells. (A,C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B,D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000 ×, D: M = 4,000 ×. (E,F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light gray shade compared to flat cell regions in F, appearing in a darker gray, E: M = 100,000×, F: M = 120,000×.
Figure Legend Snippet: Correlative FM and STEM of whole breast cancer cells. (A,C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B,D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000 ×, D: M = 4,000 ×. (E,F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light gray shade compared to flat cell regions in F, appearing in a darker gray, E: M = 100,000×, F: M = 120,000×.

Techniques Used: Fluorescence, Labeling

Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. (B) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. (D) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).
Figure Legend Snippet: Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. (B) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. (D) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).

Techniques Used: Transduction, Construct, Labeling, Evaporation, Microscopy, Fluorescence, Transmission Assay, Electron Microscopy

Correlative FM and STEM of whole breast cancer cells treated with Cytochalasin D. (A) Cropped fluorescence micrograph of Cytochalasin D treated SKBR3 cells with actin-GFP in green, QD-labeled membrane ErbB2 in red and overlapping areas in yellow. (B,C) Corresponding STEM images of graphene-covered SKBR3 breast cancer cells ( M = B: 1000×, C: 30,000×). (D) Cytochalasin D inducedindentations are marked exemplarily with white arrowheads. STEM image of region enclosed with dashed square in A ( M = 100,000×). Automatically detected QD-labels are marked in yellow to enhance visibility. (E) g ( r ) of QD-labels collected in seven images of cell edges of Cytochalasin D treated cells. The arrow marks a peak at r = 36 nm, and the dotted line indicates r = 20 nm. g ( r ) of simulated data of randomly positioned labels.
Figure Legend Snippet: Correlative FM and STEM of whole breast cancer cells treated with Cytochalasin D. (A) Cropped fluorescence micrograph of Cytochalasin D treated SKBR3 cells with actin-GFP in green, QD-labeled membrane ErbB2 in red and overlapping areas in yellow. (B,C) Corresponding STEM images of graphene-covered SKBR3 breast cancer cells ( M = B: 1000×, C: 30,000×). (D) Cytochalasin D inducedindentations are marked exemplarily with white arrowheads. STEM image of region enclosed with dashed square in A ( M = 100,000×). Automatically detected QD-labels are marked in yellow to enhance visibility. (E) g ( r ) of QD-labels collected in seven images of cell edges of Cytochalasin D treated cells. The arrow marks a peak at r = 36 nm, and the dotted line indicates r = 20 nm. g ( r ) of simulated data of randomly positioned labels.

Techniques Used: Fluorescence, Labeling

Correlative FM and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M = 30,000×. (D) Region selected from C imaged at M = 100,000×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180 ± 30°.
Figure Legend Snippet: Correlative FM and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M = 30,000×. (D) Region selected from C imaged at M = 100,000×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180 ± 30°.

Techniques Used: Labeling, Fluorescence

10) Product Images from "Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin"

Article Title: Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

Journal: bioRxiv

doi: 10.1101/2020.01.14.906040

Correlative fluorescence microscopy and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M =30,000×. (D) Region selected from C imaged at M =100,000 ×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180° ± 30°.
Figure Legend Snippet: Correlative fluorescence microscopy and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M =30,000×. (D) Region selected from C imaged at M =100,000 ×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180° ± 30°.

Techniques Used: Fluorescence, Microscopy, Labeling

STEM of whole breast cancer cells treated with Cytochalasin D. (A) STEM of graphene-covered SKBR3 breast cancer cells ( M = 30,000×). Cytochalasin D induced-fingerlike protrusions are marked exemplarily with white arrowheads. (B) STEM image of region enclosed with dashed square in A ( M =100,000×). Automatically detected QD-labels are marked in yellow to enhance visibility. (C) g ( r ) of QD-labels collected in 7 images of cell edges of Cytochalasin D treated cells. The arrow marks a peak at r = 36 nm, and the dotted line indicates r = 20 nm. g ( r ) of simulated data of randomly positioned labels.
Figure Legend Snippet: STEM of whole breast cancer cells treated with Cytochalasin D. (A) STEM of graphene-covered SKBR3 breast cancer cells ( M = 30,000×). Cytochalasin D induced-fingerlike protrusions are marked exemplarily with white arrowheads. (B) STEM image of region enclosed with dashed square in A ( M =100,000×). Automatically detected QD-labels are marked in yellow to enhance visibility. (C) g ( r ) of QD-labels collected in 7 images of cell edges of Cytochalasin D treated cells. The arrow marks a peak at r = 36 nm, and the dotted line indicates r = 20 nm. g ( r ) of simulated data of randomly positioned labels.

Techniques Used:

Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. ( B ) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. ( D ) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).
Figure Legend Snippet: Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. ( B ) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. ( D ) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).

Techniques Used: Transduction, Construct, Labeling, Evaporation, Microscopy, Fluorescence, Transmission Assay, Electron Microscopy

Correlative fluorescence microscopy and STEM of whole breast cancer cells. (A, C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B, D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000×, D: M = 4,000×. (E, F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light grey shade compared to flat cell regions in F, appearing in a darker grey, E: M = 100,000×, F: M = 120,000×.
Figure Legend Snippet: Correlative fluorescence microscopy and STEM of whole breast cancer cells. (A, C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B, D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000×, D: M = 4,000×. (E, F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light grey shade compared to flat cell regions in F, appearing in a darker grey, E: M = 100,000×, F: M = 120,000×.

Techniques Used: Fluorescence, Microscopy, Labeling

11) Product Images from "Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin"

Article Title: Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

Journal: bioRxiv

doi: 10.1101/2020.01.14.906040

Correlative fluorescence microscopy and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M =30,000×. (D) Region selected from C imaged at M =100,000 ×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180° ± 30°.
Figure Legend Snippet: Correlative fluorescence microscopy and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M =30,000×. (D) Region selected from C imaged at M =100,000 ×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180° ± 30°.

Techniques Used: Fluorescence, Microscopy, Labeling

Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. ( B ) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. ( D ) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).
Figure Legend Snippet: Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. ( B ) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. ( D ) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).

Techniques Used: Transduction, Construct, Labeling, Evaporation, Microscopy, Fluorescence, Transmission Assay, Electron Microscopy

Correlative fluorescence microscopy and STEM of whole breast cancer cells. (A, C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B, D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000×, D: M = 4,000×. (E, F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light grey shade compared to flat cell regions in F, appearing in a darker grey, E: M = 100,000×, F: M = 120,000×.
Figure Legend Snippet: Correlative fluorescence microscopy and STEM of whole breast cancer cells. (A, C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B, D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000×, D: M = 4,000×. (E, F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light grey shade compared to flat cell regions in F, appearing in a darker grey, E: M = 100,000×, F: M = 120,000×.

Techniques Used: Fluorescence, Microscopy, Labeling

12) Product Images from "Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding"

Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074200

Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.
Figure Legend Snippet: Fluorescence intensity measured from confocal microscopy images of T47D cells labeled with 50-conjugated EGFR affibody, and a mixture of 25 nM dye-conjugated affibody and 25 nM unlabeled affibody. Three dyes were selected to cover the range of mobilities (Alexa 488, high mobility; CF 633, moderate mobility; Atto 565, low mobility). Columns represent the median of the distribution of membrane region pixel intensities derived from at least 100 cells. Error bars represent the positions of the 1 st and 3 rd quartile of the distributions.

Techniques Used: Fluorescence, Confocal Microscopy, Labeling, Derivative Assay

13) Product Images from "Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake"

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

Journal: EJNMMI Research

doi: 10.1186/s13550-016-0213-8

PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

Techniques Used: Positron Emission Tomography

PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

Techniques Used: Positron Emission Tomography

14) Product Images from "Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents"

Article Title: Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents

Journal: Nature Communications

doi: 10.1038/ncomms15623

Analysis of angiopep-2 transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.
Figure Legend Snippet: Analysis of angiopep-2 transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.

Techniques Used: Fluorescence, Expressing, Staining, Incubation, Concentration Assay, Confocal Microscopy, Permeability, Injection, Mouse Assay

Analysis of angiopep-2 transport using the well-established in vitro BBB Transwell system. Permeability assay using the BBB co-culture Transwell model showing that the ( a ) scrambled control and ( b ) angiopep-2 displayed significantly lower permeation in the co-culture model compared to inserts containing no cells (which represent passive diffusion). ( c ) The Transwell co-culture model failed to differentiate between the permeability of angiopep-2 and the scrambled peptide. For all permeability assays, TAMRA-labelled angiopep-2 (or scramble) peptide (10 μM concentration) was added onto the apical side of the Transwells of the co-culture model after 84 h of incubation. The basal side of the Transwell was imaged using fluorescence microscopy, and the fluorescence intensity was quantified over 40 h. The plots show the accumulation of fluorescence intensity over time with s.d. error bars ( n transwell =2, n experiment =2). Statistical analysis was performed using the one-way ANOVA and Tukey's multiple comparison test. ( d ) Confocal images showing higher expression of ZO-1 (tight junction), P-gp (efflux pump) and β-catenin (adherens junction; shown in white) on the surface of BBB spheroids compared with hCMEC/D3 ECs in the triple co-culture Transwell model after 48 h. Cell nuclei were labelled with Hoechst dye (shown in blue). Scale bar, 100 μm.
Figure Legend Snippet: Analysis of angiopep-2 transport using the well-established in vitro BBB Transwell system. Permeability assay using the BBB co-culture Transwell model showing that the ( a ) scrambled control and ( b ) angiopep-2 displayed significantly lower permeation in the co-culture model compared to inserts containing no cells (which represent passive diffusion). ( c ) The Transwell co-culture model failed to differentiate between the permeability of angiopep-2 and the scrambled peptide. For all permeability assays, TAMRA-labelled angiopep-2 (or scramble) peptide (10 μM concentration) was added onto the apical side of the Transwells of the co-culture model after 84 h of incubation. The basal side of the Transwell was imaged using fluorescence microscopy, and the fluorescence intensity was quantified over 40 h. The plots show the accumulation of fluorescence intensity over time with s.d. error bars ( n transwell =2, n experiment =2). Statistical analysis was performed using the one-way ANOVA and Tukey's multiple comparison test. ( d ) Confocal images showing higher expression of ZO-1 (tight junction), P-gp (efflux pump) and β-catenin (adherens junction; shown in white) on the surface of BBB spheroids compared with hCMEC/D3 ECs in the triple co-culture Transwell model after 48 h. Cell nuclei were labelled with Hoechst dye (shown in blue). Scale bar, 100 μm.

Techniques Used: In Vitro, Permeability, Co-Culture Assay, Diffusion-based Assay, Concentration Assay, Incubation, Fluorescence, Microscopy, Expressing

15) Product Images from "Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin"

Article Title: Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00521

Correlative FM and STEM of whole breast cancer cells. (A,C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B,D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000 ×, D: M = 4,000 ×. (E,F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light gray shade compared to flat cell regions in F, appearing in a darker gray, E: M = 100,000×, F: M = 120,000×.
Figure Legend Snippet: Correlative FM and STEM of whole breast cancer cells. (A,C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B,D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000 ×, D: M = 4,000 ×. (E,F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light gray shade compared to flat cell regions in F, appearing in a darker gray, E: M = 100,000×, F: M = 120,000×.

Techniques Used: Fluorescence, Labeling

Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. (B) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. (D) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).
Figure Legend Snippet: Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. (B) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. (D) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).

Techniques Used: Transduction, Construct, Labeling, Evaporation, Microscopy, Fluorescence, Transmission Assay, Electron Microscopy

Correlative FM and STEM of whole breast cancer cells treated with Cytochalasin D. (A) Cropped fluorescence micrograph of Cytochalasin D treated SKBR3 cells with actin-GFP in green, QD-labeled membrane ErbB2 in red and overlapping areas in yellow. (B,C) Corresponding STEM images of graphene-covered SKBR3 breast cancer cells ( M = B: 1000×, C: 30,000×). (D) Cytochalasin D inducedindentations are marked exemplarily with white arrowheads. STEM image of region enclosed with dashed square in A ( M = 100,000×). Automatically detected QD-labels are marked in yellow to enhance visibility. (E) g ( r ) of QD-labels collected in seven images of cell edges of Cytochalasin D treated cells. The arrow marks a peak at r = 36 nm, and the dotted line indicates r = 20 nm. g ( r ) of simulated data of randomly positioned labels.
Figure Legend Snippet: Correlative FM and STEM of whole breast cancer cells treated with Cytochalasin D. (A) Cropped fluorescence micrograph of Cytochalasin D treated SKBR3 cells with actin-GFP in green, QD-labeled membrane ErbB2 in red and overlapping areas in yellow. (B,C) Corresponding STEM images of graphene-covered SKBR3 breast cancer cells ( M = B: 1000×, C: 30,000×). (D) Cytochalasin D inducedindentations are marked exemplarily with white arrowheads. STEM image of region enclosed with dashed square in A ( M = 100,000×). Automatically detected QD-labels are marked in yellow to enhance visibility. (E) g ( r ) of QD-labels collected in seven images of cell edges of Cytochalasin D treated cells. The arrow marks a peak at r = 36 nm, and the dotted line indicates r = 20 nm. g ( r ) of simulated data of randomly positioned labels.

Techniques Used: Fluorescence, Labeling

Correlative FM and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M = 30,000×. (D) Region selected from C imaged at M = 100,000×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180 ± 30°.
Figure Legend Snippet: Correlative FM and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M = 30,000×. (D) Region selected from C imaged at M = 100,000×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180 ± 30°.

Techniques Used: Labeling, Fluorescence

16) Product Images from "Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents"

Article Title: Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents

Journal: Nature Communications

doi: 10.1038/ncomms15623

Analysis of angiopep-2 transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.
Figure Legend Snippet: Analysis of angiopep-2 transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.

Techniques Used: Fluorescence, Expressing, Staining, Incubation, Concentration Assay, Confocal Microscopy, Permeability, Injection, Mouse Assay

Analysis of angiopep-2 transport using the well-established in vitro BBB Transwell system. Permeability assay using the BBB co-culture Transwell model showing that the ( a ) scrambled control and ( b ) angiopep-2 displayed significantly lower permeation in the co-culture model compared to inserts containing no cells (which represent passive diffusion). ( c ) The Transwell co-culture model failed to differentiate between the permeability of angiopep-2 and the scrambled peptide. For all permeability assays, TAMRA-labelled angiopep-2 (or scramble) peptide (10 μM concentration) was added onto the apical side of the Transwells of the co-culture model after 84 h of incubation. The basal side of the Transwell was imaged using fluorescence microscopy, and the fluorescence intensity was quantified over 40 h. The plots show the accumulation of fluorescence intensity over time with s.d. error bars ( n transwell =2, n experiment =2). Statistical analysis was performed using the one-way ANOVA and Tukey's multiple comparison test. ( d ) Confocal images showing higher expression of ZO-1 (tight junction), P-gp (efflux pump) and β-catenin (adherens junction; shown in white) on the surface of BBB spheroids compared with hCMEC/D3 ECs in the triple co-culture Transwell model after 48 h. Cell nuclei were labelled with Hoechst dye (shown in blue). Scale bar, 100 μm.
Figure Legend Snippet: Analysis of angiopep-2 transport using the well-established in vitro BBB Transwell system. Permeability assay using the BBB co-culture Transwell model showing that the ( a ) scrambled control and ( b ) angiopep-2 displayed significantly lower permeation in the co-culture model compared to inserts containing no cells (which represent passive diffusion). ( c ) The Transwell co-culture model failed to differentiate between the permeability of angiopep-2 and the scrambled peptide. For all permeability assays, TAMRA-labelled angiopep-2 (or scramble) peptide (10 μM concentration) was added onto the apical side of the Transwells of the co-culture model after 84 h of incubation. The basal side of the Transwell was imaged using fluorescence microscopy, and the fluorescence intensity was quantified over 40 h. The plots show the accumulation of fluorescence intensity over time with s.d. error bars ( n transwell =2, n experiment =2). Statistical analysis was performed using the one-way ANOVA and Tukey's multiple comparison test. ( d ) Confocal images showing higher expression of ZO-1 (tight junction), P-gp (efflux pump) and β-catenin (adherens junction; shown in white) on the surface of BBB spheroids compared with hCMEC/D3 ECs in the triple co-culture Transwell model after 48 h. Cell nuclei were labelled with Hoechst dye (shown in blue). Scale bar, 100 μm.

Techniques Used: In Vitro, Permeability, Co-Culture Assay, Diffusion-based Assay, Concentration Assay, Incubation, Fluorescence, Microscopy, Expressing

17) Product Images from "Fluorescent Affibody Peptide Penetration in Glioma Margin Is Superior to Full Antibody"

Article Title: Fluorescent Affibody Peptide Penetration in Glioma Margin Is Superior to Full Antibody

Journal: PLoS ONE

doi: 10.1371/journal.pone.0060390

Examination of autofluorescence at both Affibdoy and cetuximab channels over the tumor region Tumor outlined by GFP signal (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Area enclosed in the yellow box is shown at 20 times magnification in (C). Autofluorescence at both the cetuximab channel (D) and the Affibody channel (E) show significant contrast between tumor and non-tumor regions with autofluorescence greatest at the tumor center. No significant change between tumor interior, tumor edge and non-tumor area is seen for the fraction of signal at the Affibody channel (F).
Figure Legend Snippet: Examination of autofluorescence at both Affibdoy and cetuximab channels over the tumor region Tumor outlined by GFP signal (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Area enclosed in the yellow box is shown at 20 times magnification in (C). Autofluorescence at both the cetuximab channel (D) and the Affibody channel (E) show significant contrast between tumor and non-tumor regions with autofluorescence greatest at the tumor center. No significant change between tumor interior, tumor edge and non-tumor area is seen for the fraction of signal at the Affibody channel (F).

Techniques Used: Staining

Comparison of raw fluorescent signals. Raw fluorescent signals from various regions shown at both cetuximab channel (left) and Affibody channel (right) using box and whisker plots. Signal from injected animals are offset to the left while those from non-injected control animals are offset to the right with boxes shaded. The central lines are the medians, the edges of the boxes are the 25th and 75th percentiles and individual data points are plotted as open circles.
Figure Legend Snippet: Comparison of raw fluorescent signals. Raw fluorescent signals from various regions shown at both cetuximab channel (left) and Affibody channel (right) using box and whisker plots. Signal from injected animals are offset to the left while those from non-injected control animals are offset to the right with boxes shaded. The central lines are the medians, the edges of the boxes are the 25th and 75th percentiles and individual data points are plotted as open circles.

Techniques Used: Whisker Assay, Injection

Examination of Affibody and cetuximab distribution over the tumor region. Signal from GFP outlines the tumor (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Tumor is outlined in red and area enclosed in the yellow box is shown at 20 times magnification in (C). Fluorescent signal at cetuximab channel shows significant contrast in much of the tumor, but appears reduced around the edges (D). Fluorescent signal at Affibody channel shows significant contrast in the tumor and over a broader region of the tumor (E). Fraction of signal from the Affibody channel is shown in (F) and demonstrates significant deviation in signal from the two channels at the edge and interior of the tumor.
Figure Legend Snippet: Examination of Affibody and cetuximab distribution over the tumor region. Signal from GFP outlines the tumor (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Tumor is outlined in red and area enclosed in the yellow box is shown at 20 times magnification in (C). Fluorescent signal at cetuximab channel shows significant contrast in much of the tumor, but appears reduced around the edges (D). Fluorescent signal at Affibody channel shows significant contrast in the tumor and over a broader region of the tumor (E). Fraction of signal from the Affibody channel is shown in (F) and demonstrates significant deviation in signal from the two channels at the edge and interior of the tumor.

Techniques Used: Staining

Comparison of plasma excretion for the two proteins. Plasma excretion data with error bars and bi-exponential curve fits to the data are shown for cetuximab-IRDye 680RD (left) and Affibody-IRDye 800CW (right). Curve fit equations are also shown where FL is fluorescence intensity. R-squared values of 0.71 and 0.90 for cetuximab and Affibody fits respectively.
Figure Legend Snippet: Comparison of plasma excretion for the two proteins. Plasma excretion data with error bars and bi-exponential curve fits to the data are shown for cetuximab-IRDye 680RD (left) and Affibody-IRDye 800CW (right). Curve fit equations are also shown where FL is fluorescence intensity. R-squared values of 0.71 and 0.90 for cetuximab and Affibody fits respectively.

Techniques Used: Fluorescence

18) Product Images from "Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake"

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

Journal: EJNMMI Research

doi: 10.1186/s13550-016-0213-8

PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

Techniques Used: Positron Emission Tomography

PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

Techniques Used: Positron Emission Tomography

19) Product Images from "Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake"

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

Journal: EJNMMI Research

doi: 10.1186/s13550-016-0213-8

PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

Techniques Used: Positron Emission Tomography

PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

Techniques Used: Positron Emission Tomography

20) Product Images from "Targeting cancer cell-specific RNA interference by siRNA delivery using a complex carrier of affibody-displaying bio-nanocapsules and liposomes"

Article Title: Targeting cancer cell-specific RNA interference by siRNA delivery using a complex carrier of affibody-displaying bio-nanocapsules and liposomes

Journal: Journal of Nanobiotechnology

doi: 10.1186/1477-3155-11-19

Quantification of RNAi (black bars) and cell survival rates (gray bars) of HER2-positive SKBR3 and HER2-negative HeLa cells treated by siRNA combined with Z HER2 -BNC/LP (left side) and Z WT -BNC/LP (right side) (final conc. 25 nM as siRNA).
Figure Legend Snippet: Quantification of RNAi (black bars) and cell survival rates (gray bars) of HER2-positive SKBR3 and HER2-negative HeLa cells treated by siRNA combined with Z HER2 -BNC/LP (left side) and Z WT -BNC/LP (right side) (final conc. 25 nM as siRNA).

Techniques Used:

Quantification of RNAi in HER2-positive SKBR3 (A) and HER2-negative HeLa (B) cells treated by siRNA combined with RNAiMAX (white bars), LPs (gray bars) and Z HER2 -BNC/LP complex (black bars). The GFP expressions of the cells were analyzed using a flow cytometer and results are expressed as a percentage of the GFP-expressing cellular quantity in untreated controls. The x-axis represents the final concentration of siRNA in the medium adjusted to 2 ml.
Figure Legend Snippet: Quantification of RNAi in HER2-positive SKBR3 (A) and HER2-negative HeLa (B) cells treated by siRNA combined with RNAiMAX (white bars), LPs (gray bars) and Z HER2 -BNC/LP complex (black bars). The GFP expressions of the cells were analyzed using a flow cytometer and results are expressed as a percentage of the GFP-expressing cellular quantity in untreated controls. The x-axis represents the final concentration of siRNA in the medium adjusted to 2 ml.

Techniques Used: Flow Cytometry, Cytometry, Expressing, Concentration Assay

Quantification of RNAi (black bars) and cell survival rates (gray bars) of HER2-positive SKBR3 (A) and HER2-negative HeLa (B) cells treated by negative siRNA combined with RNAiMAX, LPs and Z HER2 -BNC/LP complex (final conc. 25 nM as siRNA).
Figure Legend Snippet: Quantification of RNAi (black bars) and cell survival rates (gray bars) of HER2-positive SKBR3 (A) and HER2-negative HeLa (B) cells treated by negative siRNA combined with RNAiMAX, LPs and Z HER2 -BNC/LP complex (final conc. 25 nM as siRNA).

Techniques Used:

21) Product Images from "Fluorescent Affibody Peptide Penetration in Glioma Margin Is Superior to Full Antibody"

Article Title: Fluorescent Affibody Peptide Penetration in Glioma Margin Is Superior to Full Antibody

Journal: PLoS ONE

doi: 10.1371/journal.pone.0060390

Examination of autofluorescence at both Affibdoy and cetuximab channels over the tumor region Tumor outlined by GFP signal (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Area enclosed in the yellow box is shown at 20 times magnification in (C). Autofluorescence at both the cetuximab channel (D) and the Affibody channel (E) show significant contrast between tumor and non-tumor regions with autofluorescence greatest at the tumor center. No significant change between tumor interior, tumor edge and non-tumor area is seen for the fraction of signal at the Affibody channel (F).
Figure Legend Snippet: Examination of autofluorescence at both Affibdoy and cetuximab channels over the tumor region Tumor outlined by GFP signal (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Area enclosed in the yellow box is shown at 20 times magnification in (C). Autofluorescence at both the cetuximab channel (D) and the Affibody channel (E) show significant contrast between tumor and non-tumor regions with autofluorescence greatest at the tumor center. No significant change between tumor interior, tumor edge and non-tumor area is seen for the fraction of signal at the Affibody channel (F).

Techniques Used: Staining

Comparison of raw fluorescent signals. Raw fluorescent signals from various regions shown at both cetuximab channel (left) and Affibody channel (right) using box and whisker plots. Signal from injected animals are offset to the left while those from non-injected control animals are offset to the right with boxes shaded. The central lines are the medians, the edges of the boxes are the 25th and 75th percentiles and individual data points are plotted as open circles.
Figure Legend Snippet: Comparison of raw fluorescent signals. Raw fluorescent signals from various regions shown at both cetuximab channel (left) and Affibody channel (right) using box and whisker plots. Signal from injected animals are offset to the left while those from non-injected control animals are offset to the right with boxes shaded. The central lines are the medians, the edges of the boxes are the 25th and 75th percentiles and individual data points are plotted as open circles.

Techniques Used: Whisker Assay, Injection

Examination of Affibody and cetuximab distribution over the tumor region. Signal from GFP outlines the tumor (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Tumor is outlined in red and area enclosed in the yellow box is shown at 20 times magnification in (C). Fluorescent signal at cetuximab channel shows significant contrast in much of the tumor, but appears reduced around the edges (D). Fluorescent signal at Affibody channel shows significant contrast in the tumor and over a broader region of the tumor (E). Fraction of signal from the Affibody channel is shown in (F) and demonstrates significant deviation in signal from the two channels at the edge and interior of the tumor.
Figure Legend Snippet: Examination of Affibody and cetuximab distribution over the tumor region. Signal from GFP outlines the tumor (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Tumor is outlined in red and area enclosed in the yellow box is shown at 20 times magnification in (C). Fluorescent signal at cetuximab channel shows significant contrast in much of the tumor, but appears reduced around the edges (D). Fluorescent signal at Affibody channel shows significant contrast in the tumor and over a broader region of the tumor (E). Fraction of signal from the Affibody channel is shown in (F) and demonstrates significant deviation in signal from the two channels at the edge and interior of the tumor.

Techniques Used: Staining

Comparison of plasma excretion for the two proteins. Plasma excretion data with error bars and bi-exponential curve fits to the data are shown for cetuximab-IRDye 680RD (left) and Affibody-IRDye 800CW (right). Curve fit equations are also shown where FL is fluorescence intensity. R-squared values of 0.71 and 0.90 for cetuximab and Affibody fits respectively.
Figure Legend Snippet: Comparison of plasma excretion for the two proteins. Plasma excretion data with error bars and bi-exponential curve fits to the data are shown for cetuximab-IRDye 680RD (left) and Affibody-IRDye 800CW (right). Curve fit equations are also shown where FL is fluorescence intensity. R-squared values of 0.71 and 0.90 for cetuximab and Affibody fits respectively.

Techniques Used: Fluorescence

22) Product Images from "Targeted Delivery of Deoxycytidine Kinase to Her2-Positive Cells Enhances the Efficacy of the Nucleoside Analog Fludarabine"

Article Title: Targeted Delivery of Deoxycytidine Kinase to Her2-Positive Cells Enhances the Efficacy of the Nucleoside Analog Fludarabine

Journal: PLoS ONE

doi: 10.1371/journal.pone.0157114

Strategy for preferential activation of prodrugs at target cells and the Her2-affinity reagents used in this study. (A) This strategy relies on a bi-modular fusion protein composed of a cell marker-targeting module (square labeled with T) genetically fused to an enzyme that catalyzes the activation of a prodrug (circle labeled with E). This fusion protein is administered systemically (point 1 ), but it accumulates at the targeted cells by binding to a specific cell surface protein (point 2 ). The fusion protein then enters the cell via receptor-mediated endocytosis or by membrane recycling (point 3 ). Subsequent administration of an appropriate prodrug results in its preferential activation in the targeted cells (point 4 ), thereby killing the targeted cell. (B) Ribbon diagram of the reagents with their molecular size indicated. Affibody, DARPin, and dCK models from PDB IDs 1LP1, 2JAB, and 1P5Z, respectively. The fusion proteins were modeled based on the individual structures. The dimeric nature of dCK result in molecules that contains two anti-Her2 modules, which are expected to increase its avidity to the receptor relative to the single affinity modules. Green spheres denote the substrates binding sites in dCK. (C) SDS-PAGE demonstrates the > 95% purity of the reagents. We note that the DARPin module runs as a smaller protein than the expected size. (D) Gel-filtration analysis of the reagents. The observed elution volumes correspond to the expected sizes of the reagents, with the fusion protein being dimeric, and the single affinity modules being monomeric.
Figure Legend Snippet: Strategy for preferential activation of prodrugs at target cells and the Her2-affinity reagents used in this study. (A) This strategy relies on a bi-modular fusion protein composed of a cell marker-targeting module (square labeled with T) genetically fused to an enzyme that catalyzes the activation of a prodrug (circle labeled with E). This fusion protein is administered systemically (point 1 ), but it accumulates at the targeted cells by binding to a specific cell surface protein (point 2 ). The fusion protein then enters the cell via receptor-mediated endocytosis or by membrane recycling (point 3 ). Subsequent administration of an appropriate prodrug results in its preferential activation in the targeted cells (point 4 ), thereby killing the targeted cell. (B) Ribbon diagram of the reagents with their molecular size indicated. Affibody, DARPin, and dCK models from PDB IDs 1LP1, 2JAB, and 1P5Z, respectively. The fusion proteins were modeled based on the individual structures. The dimeric nature of dCK result in molecules that contains two anti-Her2 modules, which are expected to increase its avidity to the receptor relative to the single affinity modules. Green spheres denote the substrates binding sites in dCK. (C) SDS-PAGE demonstrates the > 95% purity of the reagents. We note that the DARPin module runs as a smaller protein than the expected size. (D) Gel-filtration analysis of the reagents. The observed elution volumes correspond to the expected sizes of the reagents, with the fusion protein being dimeric, and the single affinity modules being monomeric.

Techniques Used: Activation Assay, Marker, Labeling, Binding Assay, SDS Page, Filtration

Binding of bi-modular anti-Her2 -dCK fusion proteins to cancer cells. (A) One hundred thousand cells were treated with 1 μM of Alexa Fluor ® 647 dye (red in the images) conjugated reagents and incubated for 2 h at either 4°C or 37°C. The nuclei of treated cells were stained with DAPI (blue) and visualized at 40x using a Zeiss confocal laser scanning microscope. Images are representative of three independent trials. Scale bar depicts 10 μm. Note the much-increased signal for the fusion proteins with the Her2-postive cells lines (BT-474 and SK-OV3), relative to the Her2-negative MCF-7 cell line. The stronger intracellular signal at 37°C relative to that observed at 4°C indicates that at least a fraction of the fusion proteins has internalized. (B) Internalization of DARPin-dCK in BT-474 cells. Co-localization of receptor bound reagents with intracellular vesicles was detected by treating 5x10 4 cells with 1.5x10 6 particles each of CellLight ® Reagents *BacMam 2.0* GFP markers for early and late endosomes and lysosomes, for 18 h at 37°C, according to the manufacturer’s protocol. These cells were then treated with 0.5 μM of Alexa Fluor ® 647 conjugated DARPin-dCK, as in A. Image sections at the z plane at 40x resolution of a single BT-474 cell are shown on the left panel. A high power image of a single section is shown on the right panel. GFP fluorescence indicates location of endosomes and lysosomes, while red fluorescence indicates the cellular location of DARPin-dCK. Image shown is representative of 10 cells imaged from duplicate trials. Scale bar is 10 μm.
Figure Legend Snippet: Binding of bi-modular anti-Her2 -dCK fusion proteins to cancer cells. (A) One hundred thousand cells were treated with 1 μM of Alexa Fluor ® 647 dye (red in the images) conjugated reagents and incubated for 2 h at either 4°C or 37°C. The nuclei of treated cells were stained with DAPI (blue) and visualized at 40x using a Zeiss confocal laser scanning microscope. Images are representative of three independent trials. Scale bar depicts 10 μm. Note the much-increased signal for the fusion proteins with the Her2-postive cells lines (BT-474 and SK-OV3), relative to the Her2-negative MCF-7 cell line. The stronger intracellular signal at 37°C relative to that observed at 4°C indicates that at least a fraction of the fusion proteins has internalized. (B) Internalization of DARPin-dCK in BT-474 cells. Co-localization of receptor bound reagents with intracellular vesicles was detected by treating 5x10 4 cells with 1.5x10 6 particles each of CellLight ® Reagents *BacMam 2.0* GFP markers for early and late endosomes and lysosomes, for 18 h at 37°C, according to the manufacturer’s protocol. These cells were then treated with 0.5 μM of Alexa Fluor ® 647 conjugated DARPin-dCK, as in A. Image sections at the z plane at 40x resolution of a single BT-474 cell are shown on the left panel. A high power image of a single section is shown on the right panel. GFP fluorescence indicates location of endosomes and lysosomes, while red fluorescence indicates the cellular location of DARPin-dCK. Image shown is representative of 10 cells imaged from duplicate trials. Scale bar is 10 μm.

Techniques Used: Binding Assay, Incubation, Staining, Laser-Scanning Microscopy, Fluorescence

Binding of the bi-modular fusion proteins to cancer cells measured by flow cytometry. (A) One million cells were treated with 1 μM of reagents as in Fig 2 and mean fluorescence intensity was measured using a Cyan3 fluorescent cell sorter (Beckman). Fluorescent cells were gated on cells treated with reagents that were not conjugated to dye and hence did not exhibit any fluorescence at 647 nm. Error bars correspond to standard deviations on three independent trials. (B) One million cells were treated with 1 μM dCK-AlexaFluor ™ 647 and the signal intensity was normalized to number of dye conjugations (ref 3 from Supplement). Error bars correspond to standard deviations of n = 3 trials. (C) Cell were treated with 2 μM of anti-Her2 DARPin-AlexaFluor ™ 647 and Affibody-AlexaFluor ™ 647 and measured as above. Note the much reduced mean fluorescence intensity in comparison to the fusion constructs (panel A). Error bars correspond to standard deviations of three independent trials. (D) Fluorescence intensities of cells treated with dCK-fusion protein were normalized to number of conjugated dye molecules (3), background subtracted and compared to those of cells treated with the Her2 affinity module (DARPin or affibody) alone. The resulting mean fold change in intensity was plotted for each cell type and reagent.
Figure Legend Snippet: Binding of the bi-modular fusion proteins to cancer cells measured by flow cytometry. (A) One million cells were treated with 1 μM of reagents as in Fig 2 and mean fluorescence intensity was measured using a Cyan3 fluorescent cell sorter (Beckman). Fluorescent cells were gated on cells treated with reagents that were not conjugated to dye and hence did not exhibit any fluorescence at 647 nm. Error bars correspond to standard deviations on three independent trials. (B) One million cells were treated with 1 μM dCK-AlexaFluor ™ 647 and the signal intensity was normalized to number of dye conjugations (ref 3 from Supplement). Error bars correspond to standard deviations of n = 3 trials. (C) Cell were treated with 2 μM of anti-Her2 DARPin-AlexaFluor ™ 647 and Affibody-AlexaFluor ™ 647 and measured as above. Note the much reduced mean fluorescence intensity in comparison to the fusion constructs (panel A). Error bars correspond to standard deviations of three independent trials. (D) Fluorescence intensities of cells treated with dCK-fusion protein were normalized to number of conjugated dye molecules (3), background subtracted and compared to those of cells treated with the Her2 affinity module (DARPin or affibody) alone. The resulting mean fold change in intensity was plotted for each cell type and reagent.

Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Fluorescence, Construct

23) Product Images from "Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake"

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

Journal: EJNMMI Research

doi: 10.1186/s13550-016-0213-8

PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

Techniques Used: Positron Emission Tomography

PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

Techniques Used: Positron Emission Tomography

24) Product Images from "Fluorescent Affibody Peptide Penetration in Glioma Margin Is Superior to Full Antibody"

Article Title: Fluorescent Affibody Peptide Penetration in Glioma Margin Is Superior to Full Antibody

Journal: PLoS ONE

doi: 10.1371/journal.pone.0060390

Examination of autofluorescence at both Affibdoy and cetuximab channels over the tumor region Tumor outlined by GFP signal (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Area enclosed in the yellow box is shown at 20 times magnification in (C). Autofluorescence at both the cetuximab channel (D) and the Affibody channel (E) show significant contrast between tumor and non-tumor regions with autofluorescence greatest at the tumor center. No significant change between tumor interior, tumor edge and non-tumor area is seen for the fraction of signal at the Affibody channel (F).
Figure Legend Snippet: Examination of autofluorescence at both Affibdoy and cetuximab channels over the tumor region Tumor outlined by GFP signal (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Area enclosed in the yellow box is shown at 20 times magnification in (C). Autofluorescence at both the cetuximab channel (D) and the Affibody channel (E) show significant contrast between tumor and non-tumor regions with autofluorescence greatest at the tumor center. No significant change between tumor interior, tumor edge and non-tumor area is seen for the fraction of signal at the Affibody channel (F).

Techniques Used: Staining

Comparison of raw fluorescent signals. Raw fluorescent signals from various regions shown at both cetuximab channel (left) and Affibody channel (right) using box and whisker plots. Signal from injected animals are offset to the left while those from non-injected control animals are offset to the right with boxes shaded. The central lines are the medians, the edges of the boxes are the 25th and 75th percentiles and individual data points are plotted as open circles.
Figure Legend Snippet: Comparison of raw fluorescent signals. Raw fluorescent signals from various regions shown at both cetuximab channel (left) and Affibody channel (right) using box and whisker plots. Signal from injected animals are offset to the left while those from non-injected control animals are offset to the right with boxes shaded. The central lines are the medians, the edges of the boxes are the 25th and 75th percentiles and individual data points are plotted as open circles.

Techniques Used: Whisker Assay, Injection

Examination of Affibody and cetuximab distribution over the tumor region. Signal from GFP outlines the tumor (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Tumor is outlined in red and area enclosed in the yellow box is shown at 20 times magnification in (C). Fluorescent signal at cetuximab channel shows significant contrast in much of the tumor, but appears reduced around the edges (D). Fluorescent signal at Affibody channel shows significant contrast in the tumor and over a broader region of the tumor (E). Fraction of signal from the Affibody channel is shown in (F) and demonstrates significant deviation in signal from the two channels at the edge and interior of the tumor.
Figure Legend Snippet: Examination of Affibody and cetuximab distribution over the tumor region. Signal from GFP outlines the tumor (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Tumor is outlined in red and area enclosed in the yellow box is shown at 20 times magnification in (C). Fluorescent signal at cetuximab channel shows significant contrast in much of the tumor, but appears reduced around the edges (D). Fluorescent signal at Affibody channel shows significant contrast in the tumor and over a broader region of the tumor (E). Fraction of signal from the Affibody channel is shown in (F) and demonstrates significant deviation in signal from the two channels at the edge and interior of the tumor.

Techniques Used: Staining

Comparison of plasma excretion for the two proteins. Plasma excretion data with error bars and bi-exponential curve fits to the data are shown for cetuximab-IRDye 680RD (left) and Affibody-IRDye 800CW (right). Curve fit equations are also shown where FL is fluorescence intensity. R-squared values of 0.71 and 0.90 for cetuximab and Affibody fits respectively.
Figure Legend Snippet: Comparison of plasma excretion for the two proteins. Plasma excretion data with error bars and bi-exponential curve fits to the data are shown for cetuximab-IRDye 680RD (left) and Affibody-IRDye 800CW (right). Curve fit equations are also shown where FL is fluorescence intensity. R-squared values of 0.71 and 0.90 for cetuximab and Affibody fits respectively.

Techniques Used: Fluorescence

25) Product Images from "Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors"

Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

Journal: Journal of the American Chemical Society

doi: 10.1021/jacs.8b07601

(A) Top: Composite brightfield and fluorescence microscopy images of spheroids formed from transfected 4T1 cells grown either with (right) or without (left) 15 μ g/mL cumate to induce EGFR and GFP expression. Bottom: Fluorescence images of cryotome-sectioned similarly prepared spheroids showing the distribution of fluorescence throughout the interior. All images were captured and viewed under identical settings for comparison. (B) Western blot for EGFR expression of the combined lysates of five spheroids grown either with (+) or without (−) 15 μ g/mL cumate. Image shows composite overlay of both bright field imaging for display of prestained protein ladders (L) and chemiluminescence imaging for the anti-EGFR antibody. The molecular weight of each ladder band is indicated to the right.
Figure Legend Snippet: (A) Top: Composite brightfield and fluorescence microscopy images of spheroids formed from transfected 4T1 cells grown either with (right) or without (left) 15 μ g/mL cumate to induce EGFR and GFP expression. Bottom: Fluorescence images of cryotome-sectioned similarly prepared spheroids showing the distribution of fluorescence throughout the interior. All images were captured and viewed under identical settings for comparison. (B) Western blot for EGFR expression of the combined lysates of five spheroids grown either with (+) or without (−) 15 μ g/mL cumate. Image shows composite overlay of both bright field imaging for display of prestained protein ladders (L) and chemiluminescence imaging for the anti-EGFR antibody. The molecular weight of each ladder band is indicated to the right.

Techniques Used: Fluorescence, Microscopy, Transfection, Expressing, Western Blot, Imaging, Molecular Weight

(A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.
Figure Legend Snippet: (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.

Techniques Used: Polyacrylamide Gel Electrophoresis, SDS Page, Irradiation, Produced, Labeling, Transfection, Fluorescence, Incubation

Left: Spheroids were treated with 1 μ M N23BP (top) or WT (bottom) affibody for a total of 4 h (left column) and 20 h (middle and right columns) and additionally irradiated with 365 nm light for 30 min after 3.5 h incubation (right column). These spheroids were sectioned at approximately the same depth and imaged for Rhodamine and GFP distribution. Micrographs show Rhodamine signal within each section normalized by the average GFP intensity. Scale bars are 200 μ m. Right: Five spheroids were grown and irradiated for 1 h with the indicated N23BP affibody concentration in growth media. Affibody containing media was removed, and spheroids were lysed and loaded onto a PAGE gel and probed for affibody conjugates using an anti-T7 antibody. High molecular weight bands around the expected molecular weight of EGFR were observed only when the spheroid-affibody mixtures were irradiated, indicating photo-cross-linking.
Figure Legend Snippet: Left: Spheroids were treated with 1 μ M N23BP (top) or WT (bottom) affibody for a total of 4 h (left column) and 20 h (middle and right columns) and additionally irradiated with 365 nm light for 30 min after 3.5 h incubation (right column). These spheroids were sectioned at approximately the same depth and imaged for Rhodamine and GFP distribution. Micrographs show Rhodamine signal within each section normalized by the average GFP intensity. Scale bars are 200 μ m. Right: Five spheroids were grown and irradiated for 1 h with the indicated N23BP affibody concentration in growth media. Affibody containing media was removed, and spheroids were lysed and loaded onto a PAGE gel and probed for affibody conjugates using an anti-T7 antibody. High molecular weight bands around the expected molecular weight of EGFR were observed only when the spheroid-affibody mixtures were irradiated, indicating photo-cross-linking.

Techniques Used: Irradiation, Incubation, Concentration Assay, Polyacrylamide Gel Electrophoresis, Molecular Weight

26) Product Images from "Fluorescent Affibody Peptide Penetration in Glioma Margin Is Superior to Full Antibody"

Article Title: Fluorescent Affibody Peptide Penetration in Glioma Margin Is Superior to Full Antibody

Journal: PLoS ONE

doi: 10.1371/journal.pone.0060390

Examination of autofluorescence at both Affibdoy and cetuximab channels over the tumor region Tumor outlined by GFP signal (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Area enclosed in the yellow box is shown at 20 times magnification in (C). Autofluorescence at both the cetuximab channel (D) and the Affibody channel (E) show significant contrast between tumor and non-tumor regions with autofluorescence greatest at the tumor center. No significant change between tumor interior, tumor edge and non-tumor area is seen for the fraction of signal at the Affibody channel (F).
Figure Legend Snippet: Examination of autofluorescence at both Affibdoy and cetuximab channels over the tumor region Tumor outlined by GFP signal (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Area enclosed in the yellow box is shown at 20 times magnification in (C). Autofluorescence at both the cetuximab channel (D) and the Affibody channel (E) show significant contrast between tumor and non-tumor regions with autofluorescence greatest at the tumor center. No significant change between tumor interior, tumor edge and non-tumor area is seen for the fraction of signal at the Affibody channel (F).

Techniques Used: Staining

Comparison of raw fluorescent signals. Raw fluorescent signals from various regions shown at both cetuximab channel (left) and Affibody channel (right) using box and whisker plots. Signal from injected animals are offset to the left while those from non-injected control animals are offset to the right with boxes shaded. The central lines are the medians, the edges of the boxes are the 25th and 75th percentiles and individual data points are plotted as open circles.
Figure Legend Snippet: Comparison of raw fluorescent signals. Raw fluorescent signals from various regions shown at both cetuximab channel (left) and Affibody channel (right) using box and whisker plots. Signal from injected animals are offset to the left while those from non-injected control animals are offset to the right with boxes shaded. The central lines are the medians, the edges of the boxes are the 25th and 75th percentiles and individual data points are plotted as open circles.

Techniques Used: Whisker Assay, Injection

Examination of Affibody and cetuximab distribution over the tumor region. Signal from GFP outlines the tumor (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Tumor is outlined in red and area enclosed in the yellow box is shown at 20 times magnification in (C). Fluorescent signal at cetuximab channel shows significant contrast in much of the tumor, but appears reduced around the edges (D). Fluorescent signal at Affibody channel shows significant contrast in the tumor and over a broader region of the tumor (E). Fraction of signal from the Affibody channel is shown in (F) and demonstrates significant deviation in signal from the two channels at the edge and interior of the tumor.
Figure Legend Snippet: Examination of Affibody and cetuximab distribution over the tumor region. Signal from GFP outlines the tumor (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Tumor is outlined in red and area enclosed in the yellow box is shown at 20 times magnification in (C). Fluorescent signal at cetuximab channel shows significant contrast in much of the tumor, but appears reduced around the edges (D). Fluorescent signal at Affibody channel shows significant contrast in the tumor and over a broader region of the tumor (E). Fraction of signal from the Affibody channel is shown in (F) and demonstrates significant deviation in signal from the two channels at the edge and interior of the tumor.

Techniques Used: Staining

Comparison of plasma excretion for the two proteins. Plasma excretion data with error bars and bi-exponential curve fits to the data are shown for cetuximab-IRDye 680RD (left) and Affibody-IRDye 800CW (right). Curve fit equations are also shown where FL is fluorescence intensity. R-squared values of 0.71 and 0.90 for cetuximab and Affibody fits respectively.
Figure Legend Snippet: Comparison of plasma excretion for the two proteins. Plasma excretion data with error bars and bi-exponential curve fits to the data are shown for cetuximab-IRDye 680RD (left) and Affibody-IRDye 800CW (right). Curve fit equations are also shown where FL is fluorescence intensity. R-squared values of 0.71 and 0.90 for cetuximab and Affibody fits respectively.

Techniques Used: Fluorescence

27) Product Images from "Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents"

Article Title: Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents

Journal: Nature Communications

doi: 10.1038/ncomms15623

Analysis of angiopep-2 transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.
Figure Legend Snippet: Analysis of angiopep-2 transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.

Techniques Used: Fluorescence, Expressing, Staining, Incubation, Concentration Assay, Confocal Microscopy, Permeability, Injection, Mouse Assay

Analysis of angiopep-2 transport using the well-established in vitro BBB Transwell system. Permeability assay using the BBB co-culture Transwell model showing that the ( a ) scrambled control and ( b ) angiopep-2 displayed significantly lower permeation in the co-culture model compared to inserts containing no cells (which represent passive diffusion). ( c ) The Transwell co-culture model failed to differentiate between the permeability of angiopep-2 and the scrambled peptide. For all permeability assays, TAMRA-labelled angiopep-2 (or scramble) peptide (10 μM concentration) was added onto the apical side of the Transwells of the co-culture model after 84 h of incubation. The basal side of the Transwell was imaged using fluorescence microscopy, and the fluorescence intensity was quantified over 40 h. The plots show the accumulation of fluorescence intensity over time with s.d. error bars ( n transwell =2, n experiment =2). Statistical analysis was performed using the one-way ANOVA and Tukey's multiple comparison test. ( d ) Confocal images showing higher expression of ZO-1 (tight junction), P-gp (efflux pump) and β-catenin (adherens junction; shown in white) on the surface of BBB spheroids compared with hCMEC/D3 ECs in the triple co-culture Transwell model after 48 h. Cell nuclei were labelled with Hoechst dye (shown in blue). Scale bar, 100 μm.
Figure Legend Snippet: Analysis of angiopep-2 transport using the well-established in vitro BBB Transwell system. Permeability assay using the BBB co-culture Transwell model showing that the ( a ) scrambled control and ( b ) angiopep-2 displayed significantly lower permeation in the co-culture model compared to inserts containing no cells (which represent passive diffusion). ( c ) The Transwell co-culture model failed to differentiate between the permeability of angiopep-2 and the scrambled peptide. For all permeability assays, TAMRA-labelled angiopep-2 (or scramble) peptide (10 μM concentration) was added onto the apical side of the Transwells of the co-culture model after 84 h of incubation. The basal side of the Transwell was imaged using fluorescence microscopy, and the fluorescence intensity was quantified over 40 h. The plots show the accumulation of fluorescence intensity over time with s.d. error bars ( n transwell =2, n experiment =2). Statistical analysis was performed using the one-way ANOVA and Tukey's multiple comparison test. ( d ) Confocal images showing higher expression of ZO-1 (tight junction), P-gp (efflux pump) and β-catenin (adherens junction; shown in white) on the surface of BBB spheroids compared with hCMEC/D3 ECs in the triple co-culture Transwell model after 48 h. Cell nuclei were labelled with Hoechst dye (shown in blue). Scale bar, 100 μm.

Techniques Used: In Vitro, Permeability, Co-Culture Assay, Diffusion-based Assay, Concentration Assay, Incubation, Fluorescence, Microscopy, Expressing

28) Product Images from "Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake"

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

Journal: EJNMMI Research

doi: 10.1186/s13550-016-0213-8

PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

Techniques Used: Positron Emission Tomography

PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

Techniques Used: Positron Emission Tomography

Related Articles

Labeling:

Article Title: Molecular imaging of EGFR/HER2 cancer biomarkers by protein MRI contrast agents
Article Snippet: .. HER2-specific MRI contrast enhancement was blocked by preinjection of HER2 affibody labeled with Cy5.5 at 12 h and 4 h before the injection of the protein contrast agent ( ) [ ]. .. After MRI, mouse organs were dissected for histological analysis.

Article Title: Influence of composition of cysteine-containing peptide-based chelators on biodistribution of 99mTc-labeled anti-EGFR affibody molecules
Article Snippet: .. To a set of control dishes, a 50-fold molar excess of anti-EGFR antibody, cetuximab, was added before addition of the labeled affibody molecules to saturate EGFR. .. Cells were incubated for 1 h at 37 °C with 10 nM 99m Tc-labeled affibody conjugate.

Diffusion-based Assay:

Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding
Article Snippet: .. In our previous work we used mean instantaneous diffusion coefficient (D ) values as a measure of the mobility of dye conjugates bound to receptors in the plasma membrane of T47D cells, and showed that anti-EGFR affibody conjugated to Alexa Fluor 488 exhibits similar levels of mobility when bound to EGFR in cells to endogenously labelled EGFR-GFP. .. We have now investigated the mobility of a range of dye-EGFR affibody conjugates on T47D cells, using the value of D calculated for Alexa Fluor 488-labelled affibody as a reference.

Marker:

Article Title: The role of HER2 in cancer therapy and targeted drug delivery
Article Snippet: .. Keywords: HER2 receptor, tumor marker, targeted therapy, targeted drug delivery, antibody, affibody .. Human Epidermal Growth Factor Receptor 2 (HER2), also known as ErbB2, c-erbB2 or HER2/neu, is a 185 kDa protein (p185 ) with an intracellular tyrosine kinase domain and an extracelluar ligand binding domain.

Staining:

Article Title: Targeted Delivery of Deoxycytidine Kinase to Her2-Positive Cells Enhances the Efficacy of the Nucleoside Analog Fludarabine
Article Snippet: .. As expected, we see only weak staining of the Her2-negative MCF-7 cell line, suggesting little or no binding of the DARPin-dCK and affibody-dCK proteins ( ) to cells that do not express Her2. .. The images are significantly different with the Her2-positive cell lines (BT-474-JB and SK-OV-3) that show staining at both the 4°C and 37°C.

Injection:

Article Title: Molecular imaging of EGFR/HER2 cancer biomarkers by protein MRI contrast agents
Article Snippet: .. HER2-specific MRI contrast enhancement was blocked by preinjection of HER2 affibody labeled with Cy5.5 at 12 h and 4 h before the injection of the protein contrast agent ( ) [ ]. .. After MRI, mouse organs were dissected for histological analysis.

Binding Assay:

Article Title: Targeted Delivery of Deoxycytidine Kinase to Her2-Positive Cells Enhances the Efficacy of the Nucleoside Analog Fludarabine
Article Snippet: .. As expected, we see only weak staining of the Her2-negative MCF-7 cell line, suggesting little or no binding of the DARPin-dCK and affibody-dCK proteins ( ) to cells that do not express Her2. .. The images are significantly different with the Her2-positive cell lines (BT-474-JB and SK-OV-3) that show staining at both the 4°C and 37°C.

Article Title: Fluorescent Affibody Peptide Penetration in Glioma Margin Is Superior to Full Antibody
Article Snippet: .. It should be noted that the administered doses are low enough that receptor saturation or competitive binding between the Affibody and cetuximab is not expected . .. The presumed differences in vascular permeability between the Affibody and cetuximab in the tumor interior compared to the tumor edge are in conjunction with expected differences in the extent of the breakdown of the BBB in these areas.

Magnetic Resonance Imaging:

Article Title: Molecular imaging of EGFR/HER2 cancer biomarkers by protein MRI contrast agents
Article Snippet: .. HER2-specific MRI contrast enhancement was blocked by preinjection of HER2 affibody labeled with Cy5.5 at 12 h and 4 h before the injection of the protein contrast agent ( ) [ ]. .. After MRI, mouse organs were dissected for histological analysis.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 88
    Affibody mtrx gfp
    PET transaxial images ( a , b , the colour scales are the same), histograms ( c , d ) of the heterogeneity contributions (the mean intensity deviation per distance calculated according to Eq. 2 ) and surface plots ( e , f ) of the uptake of AnxA5 and <t>mTrx-GFP</t> in a FaDu xenograft. The imaging was performed in the same animal > 2 h apart on the same day. In e and f , the X - and Y -axes represent spatial dimensions and the Z -axis is the mean tracer uptake (SUV mean )
    Mtrx Gfp, supplied by Affibody, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mtrx gfp/product/Affibody
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mtrx gfp - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    84
    Affibody actin green fluorescent protein gfp fusion protein
    Correlative FM and STEM of whole breast cancer cells. (A,C) Cropped fluorescence micrographs of <t>SKBR3</t> breast cancer cells showing cellular <t>actin-GFP</t> in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B,D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000 ×, D: M = 4,000 ×. (E,F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light gray shade compared to flat cell regions in F, appearing in a darker gray, E: M = 100,000×, F: M = 120,000×.
    Actin Green Fluorescent Protein Gfp Fusion Protein, supplied by Affibody, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/actin green fluorescent protein gfp fusion protein/product/Affibody
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    actin green fluorescent protein gfp fusion protein - by Bioz Stars, 2020-09
    84/100 stars
      Buy from Supplier

    92
    Affibody gfp egfr expressive 4t1 spheroids
    (A) Top: Composite brightfield and fluorescence microscopy images of spheroids formed from transfected <t>4T1</t> cells grown either with (right) or without (left) 15 μ g/mL cumate to induce <t>EGFR</t> and <t>GFP</t> expression. Bottom: Fluorescence images of cryotome-sectioned similarly prepared spheroids showing the distribution of fluorescence throughout the interior. All images were captured and viewed under identical settings for comparison. (B) Western blot for EGFR expression of the combined lysates of five spheroids grown either with (+) or without (−) 15 μ g/mL cumate. Image shows composite overlay of both bright field imaging for display of prestained protein ladders (L) and chemiluminescence imaging for the anti-EGFR antibody. The molecular weight of each ladder band is indicated to the right.
    Gfp Egfr Expressive 4t1 Spheroids, supplied by Affibody, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp egfr expressive 4t1 spheroids/product/Affibody
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gfp egfr expressive 4t1 spheroids - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    PET transaxial images ( a , b , the colour scales are the same), histograms ( c , d ) of the heterogeneity contributions (the mean intensity deviation per distance calculated according to Eq. 2 ) and surface plots ( e , f ) of the uptake of AnxA5 and mTrx-GFP in a FaDu xenograft. The imaging was performed in the same animal > 2 h apart on the same day. In e and f , the X - and Y -axes represent spatial dimensions and the Z -axis is the mean tracer uptake (SUV mean )

    Journal: EJNMMI Physics

    Article Title: A method for comparing intra-tumoural radioactivity uptake heterogeneity in preclinical positron emission tomography studies

    doi: 10.1186/s40658-015-0124-1

    Figure Lengend Snippet: PET transaxial images ( a , b , the colour scales are the same), histograms ( c , d ) of the heterogeneity contributions (the mean intensity deviation per distance calculated according to Eq. 2 ) and surface plots ( e , f ) of the uptake of AnxA5 and mTrx-GFP in a FaDu xenograft. The imaging was performed in the same animal > 2 h apart on the same day. In e and f , the X - and Y -axes represent spatial dimensions and the Z -axis is the mean tracer uptake (SUV mean )

    Article Snippet: For radioligands, the methyl-11 C-radiolabelled Annexin A5, [methyl-11 C]-His6 -AnxA5-ST-CH3 , hereafter denoted AnxA5 (~38 kDa), mutated-thioredoxin-green fluorescence protein [methyl-11 C]-His6 -mTrx-GFP-ST-CH3 , hereafter denoted mTrx-GFP (~40 kDa) and the Affibody™ ZHER2:342 ([methyl-11 C]-ZHER2:342 -ST-CH3 ) hereafter denoted ZHER2:342 (~7 kDa) proteins had been expressed with a C-terminus selenocysteine tag (ST) and site specifically labelled with a positron-emitting carbon-11 (11 C) (t 1/2 ≈ 20 min) methyl group (CH3 ).

    Techniques: Positron Emission Tomography, Imaging

    Correlative FM and STEM of whole breast cancer cells. (A,C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B,D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000 ×, D: M = 4,000 ×. (E,F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light gray shade compared to flat cell regions in F, appearing in a darker gray, E: M = 100,000×, F: M = 120,000×.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

    doi: 10.3389/fcell.2020.00521

    Figure Lengend Snippet: Correlative FM and STEM of whole breast cancer cells. (A,C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B,D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000 ×, D: M = 4,000 ×. (E,F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light gray shade compared to flat cell regions in F, appearing in a darker gray, E: M = 100,000×, F: M = 120,000×.

    Article Snippet: SKBR3 breast cancer cells were grown on microchips, transformed to express an actin-green fluorescent protein (GFP) fusion protein, and ErbB2 was labeled via an Affibody in a two-step procedure with a quantum dot (QD) nanoparticle.

    Techniques: Fluorescence, Labeling

    Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. (B) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. (D) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

    doi: 10.3389/fcell.2020.00521

    Figure Lengend Snippet: Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. (B) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. (D) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).

    Article Snippet: SKBR3 breast cancer cells were grown on microchips, transformed to express an actin-green fluorescent protein (GFP) fusion protein, and ErbB2 was labeled via an Affibody in a two-step procedure with a quantum dot (QD) nanoparticle.

    Techniques: Transduction, Construct, Labeling, Evaporation, Microscopy, Fluorescence, Transmission Assay, Electron Microscopy

    Correlative FM and STEM of whole breast cancer cells treated with Cytochalasin D. (A) Cropped fluorescence micrograph of Cytochalasin D treated SKBR3 cells with actin-GFP in green, QD-labeled membrane ErbB2 in red and overlapping areas in yellow. (B,C) Corresponding STEM images of graphene-covered SKBR3 breast cancer cells ( M = B: 1000×, C: 30,000×). (D) Cytochalasin D inducedindentations are marked exemplarily with white arrowheads. STEM image of region enclosed with dashed square in A ( M = 100,000×). Automatically detected QD-labels are marked in yellow to enhance visibility. (E) g ( r ) of QD-labels collected in seven images of cell edges of Cytochalasin D treated cells. The arrow marks a peak at r = 36 nm, and the dotted line indicates r = 20 nm. g ( r ) of simulated data of randomly positioned labels.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

    doi: 10.3389/fcell.2020.00521

    Figure Lengend Snippet: Correlative FM and STEM of whole breast cancer cells treated with Cytochalasin D. (A) Cropped fluorescence micrograph of Cytochalasin D treated SKBR3 cells with actin-GFP in green, QD-labeled membrane ErbB2 in red and overlapping areas in yellow. (B,C) Corresponding STEM images of graphene-covered SKBR3 breast cancer cells ( M = B: 1000×, C: 30,000×). (D) Cytochalasin D inducedindentations are marked exemplarily with white arrowheads. STEM image of region enclosed with dashed square in A ( M = 100,000×). Automatically detected QD-labels are marked in yellow to enhance visibility. (E) g ( r ) of QD-labels collected in seven images of cell edges of Cytochalasin D treated cells. The arrow marks a peak at r = 36 nm, and the dotted line indicates r = 20 nm. g ( r ) of simulated data of randomly positioned labels.

    Article Snippet: SKBR3 breast cancer cells were grown on microchips, transformed to express an actin-green fluorescent protein (GFP) fusion protein, and ErbB2 was labeled via an Affibody in a two-step procedure with a quantum dot (QD) nanoparticle.

    Techniques: Fluorescence, Labeling

    Correlative FM and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M = 30,000×. (D) Region selected from C imaged at M = 100,000×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180 ± 30°.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

    doi: 10.3389/fcell.2020.00521

    Figure Lengend Snippet: Correlative FM and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M = 30,000×. (D) Region selected from C imaged at M = 100,000×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180 ± 30°.

    Article Snippet: SKBR3 breast cancer cells were grown on microchips, transformed to express an actin-green fluorescent protein (GFP) fusion protein, and ErbB2 was labeled via an Affibody in a two-step procedure with a quantum dot (QD) nanoparticle.

    Techniques: Labeling, Fluorescence

    Correlative fluorescence microscopy and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M =30,000×. (D) Region selected from C imaged at M =100,000 ×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180° ± 30°.

    Journal: bioRxiv

    Article Title: Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

    doi: 10.1101/2020.01.14.906040

    Figure Lengend Snippet: Correlative fluorescence microscopy and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M =30,000×. (D) Region selected from C imaged at M =100,000 ×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180° ± 30°.

    Article Snippet: SKBR3 breast cancer cells were grown on microchips, transformed to express an actin-green fluorescent protein (GFP) fusion protein, and ErbB2 was labeled via an Affibody in a two-step procedure with a quantum dot (QD) nanoparticle.

    Techniques: Fluorescence, Microscopy, Labeling

    Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. ( B ) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. ( D ) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).

    Journal: bioRxiv

    Article Title: Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

    doi: 10.1101/2020.01.14.906040

    Figure Lengend Snippet: Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. ( B ) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. ( D ) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).

    Article Snippet: SKBR3 breast cancer cells were grown on microchips, transformed to express an actin-green fluorescent protein (GFP) fusion protein, and ErbB2 was labeled via an Affibody in a two-step procedure with a quantum dot (QD) nanoparticle.

    Techniques: Transduction, Construct, Labeling, Evaporation, Microscopy, Fluorescence, Transmission Assay, Electron Microscopy

    Correlative fluorescence microscopy and STEM of whole breast cancer cells. (A, C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B, D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000×, D: M = 4,000×. (E, F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light grey shade compared to flat cell regions in F, appearing in a darker grey, E: M = 100,000×, F: M = 120,000×.

    Journal: bioRxiv

    Article Title: Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

    doi: 10.1101/2020.01.14.906040

    Figure Lengend Snippet: Correlative fluorescence microscopy and STEM of whole breast cancer cells. (A, C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B, D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000×, D: M = 4,000×. (E, F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light grey shade compared to flat cell regions in F, appearing in a darker grey, E: M = 100,000×, F: M = 120,000×.

    Article Snippet: SKBR3 breast cancer cells were grown on microchips, transformed to express an actin-green fluorescent protein (GFP) fusion protein, and ErbB2 was labeled via an Affibody in a two-step procedure with a quantum dot (QD) nanoparticle.

    Techniques: Fluorescence, Microscopy, Labeling

    (A) Top: Composite brightfield and fluorescence microscopy images of spheroids formed from transfected 4T1 cells grown either with (right) or without (left) 15 μ g/mL cumate to induce EGFR and GFP expression. Bottom: Fluorescence images of cryotome-sectioned similarly prepared spheroids showing the distribution of fluorescence throughout the interior. All images were captured and viewed under identical settings for comparison. (B) Western blot for EGFR expression of the combined lysates of five spheroids grown either with (+) or without (−) 15 μ g/mL cumate. Image shows composite overlay of both bright field imaging for display of prestained protein ladders (L) and chemiluminescence imaging for the anti-EGFR antibody. The molecular weight of each ladder band is indicated to the right.

    Journal: Journal of the American Chemical Society

    Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

    doi: 10.1021/jacs.8b07601

    Figure Lengend Snippet: (A) Top: Composite brightfield and fluorescence microscopy images of spheroids formed from transfected 4T1 cells grown either with (right) or without (left) 15 μ g/mL cumate to induce EGFR and GFP expression. Bottom: Fluorescence images of cryotome-sectioned similarly prepared spheroids showing the distribution of fluorescence throughout the interior. All images were captured and viewed under identical settings for comparison. (B) Western blot for EGFR expression of the combined lysates of five spheroids grown either with (+) or without (−) 15 μ g/mL cumate. Image shows composite overlay of both bright field imaging for display of prestained protein ladders (L) and chemiluminescence imaging for the anti-EGFR antibody. The molecular weight of each ladder band is indicated to the right.

    Article Snippet: GFP-EGFR expressive 4T1 spheroids in a 96-well plate were mixed with either 1 μ M Rh-conjugated N23C-BP or Rh-WT affibody and 100 μ g/mL of UCNP.

    Techniques: Fluorescence, Microscopy, Transfection, Expressing, Western Blot, Imaging, Molecular Weight

    (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.

    Journal: Journal of the American Chemical Society

    Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

    doi: 10.1021/jacs.8b07601

    Figure Lengend Snippet: (A) Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showing photo-cross-linking products of either N23BP or WT to EGFR, with or without irradiation at 980 nm. Note that only 980 nm irradiation of N23BP produced a photoproduct significantly different than free EGFR (right lane). Ladder proteins are (top to bottom) 100, 75, and 50 kDa. (B) Retention of fluorescently labeled affibodies (left, N23BP; right, WT) in 3D tumor spheroids grown from transfected 4T1 cells either induced with 15 μ g/mL cumate. Concentrations of retained affibodies were calculated by comparing spheroid lysate fluorescence to standard curves prepared for each labeled affibody. “20 IR” designates that this sample was irradiated at 980 nm after 3 h incubation, then left to grow to a total of 20 h. Lanes without an IR designation were not irradiated.

    Article Snippet: GFP-EGFR expressive 4T1 spheroids in a 96-well plate were mixed with either 1 μ M Rh-conjugated N23C-BP or Rh-WT affibody and 100 μ g/mL of UCNP.

    Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Irradiation, Produced, Labeling, Transfection, Fluorescence, Incubation

    Left: Spheroids were treated with 1 μ M N23BP (top) or WT (bottom) affibody for a total of 4 h (left column) and 20 h (middle and right columns) and additionally irradiated with 365 nm light for 30 min after 3.5 h incubation (right column). These spheroids were sectioned at approximately the same depth and imaged for Rhodamine and GFP distribution. Micrographs show Rhodamine signal within each section normalized by the average GFP intensity. Scale bars are 200 μ m. Right: Five spheroids were grown and irradiated for 1 h with the indicated N23BP affibody concentration in growth media. Affibody containing media was removed, and spheroids were lysed and loaded onto a PAGE gel and probed for affibody conjugates using an anti-T7 antibody. High molecular weight bands around the expected molecular weight of EGFR were observed only when the spheroid-affibody mixtures were irradiated, indicating photo-cross-linking.

    Journal: Journal of the American Chemical Society

    Article Title: Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

    doi: 10.1021/jacs.8b07601

    Figure Lengend Snippet: Left: Spheroids were treated with 1 μ M N23BP (top) or WT (bottom) affibody for a total of 4 h (left column) and 20 h (middle and right columns) and additionally irradiated with 365 nm light for 30 min after 3.5 h incubation (right column). These spheroids were sectioned at approximately the same depth and imaged for Rhodamine and GFP distribution. Micrographs show Rhodamine signal within each section normalized by the average GFP intensity. Scale bars are 200 μ m. Right: Five spheroids were grown and irradiated for 1 h with the indicated N23BP affibody concentration in growth media. Affibody containing media was removed, and spheroids were lysed and loaded onto a PAGE gel and probed for affibody conjugates using an anti-T7 antibody. High molecular weight bands around the expected molecular weight of EGFR were observed only when the spheroid-affibody mixtures were irradiated, indicating photo-cross-linking.

    Article Snippet: GFP-EGFR expressive 4T1 spheroids in a 96-well plate were mixed with either 1 μ M Rh-conjugated N23C-BP or Rh-WT affibody and 100 μ g/mL of UCNP.

    Techniques: Irradiation, Incubation, Concentration Assay, Polyacrylamide Gel Electrophoresis, Molecular Weight