pa25031  (GE Healthcare)

Journal: International journal of molecular sciences

Article Title: A Multifunctionalized Potyvirus-Derived Nanoparticle That Targets and Internalizes into Cancer Cells.

doi: 10.3390/ijms25084327

Figure Lengend Snippet: Figure 5. (A) Fluorescence values of the different functionalized VLPs obtained for the excitation and emission spectra corresponding to the Cy5.5 dye. (B) ELISA results using anti-cetuximab (yellow) and anti-potyvirus (pink) as primary antibodies with the different functionalized VLPs. VLP-Cy5.5 was analyzed only with anti-potyvirus. The error bars show the 95% confidence interval for the absorbance values. Functionalization A: double-functionalized VLP in which cetuximab was conjugated first and Cy5.5 afterwards. Functionalization B: double-functionalized VLP in which Cy5.5 was conjugated first and cetuximab afterwards. VLP: virus-like particle.

Article Snippet: On the other hand, the functionalization with the fluorescent dye took place by mixing 32 μg of purified VLP–Z domain (with and without previously linked cetuximab) with 3× molar excess of AmershamTM Cy5.5 mono NHS ester (GE Healthcare, Buckinghamshire, UK) to a final volume of 800 μL of HEPES 10 mM.

Techniques: Fluorescence, Enzyme-linked Immunosorbent Assay, Virus

Journal: International journal of molecular sciences

Article Title: A Multifunctionalized Potyvirus-Derived Nanoparticle That Targets and Internalizes into Cancer Cells.

doi: 10.3390/ijms25084327

Figure Lengend Snippet: Figure 6. Flow cytometry analysis of VLP–cetuximab–Cy5.5 complex binding to Cal33 (EGFR+) and THP1 (EGFR-) cells. Upper row shows Cal33 cells in the absence of VLPs (left) and in the presence of VLPs (functionalization A, (middle)), (functionalization B, (right)). The shift in the curve observed in VLPs from functionalization A with Cal33 (upper center panel) corresponds with cells binding fluorescent protein. Functionalization A: double-functionalized VLP in which cetuximab was conjugated first and Cy5.5 afterwards. Functionalization B: double-functionalized VLP in which Cy5.5 was conjugated first and cetuximab afterwards. VLP: virus-like particle.

Article Snippet: On the other hand, the functionalization with the fluorescent dye took place by mixing 32 μg of purified VLP–Z domain (with and without previously linked cetuximab) with 3× molar excess of AmershamTM Cy5.5 mono NHS ester (GE Healthcare, Buckinghamshire, UK) to a final volume of 800 μL of HEPES 10 mM.

Techniques: Flow Cytometry, Binding Assay, Virus

Journal: International journal of molecular sciences

Article Title: A Multifunctionalized Potyvirus-Derived Nanoparticle That Targets and Internalizes into Cancer Cells.

doi: 10.3390/ijms25084327

Figure Lengend Snippet: Figure 7. Representative images of Cal33 cells marked with fluorescent TuMV VLPs (either VLP-Cy5.5 alone or VLP-cetuximab-Cy5.5; in green) after an incubation of 3 h (upper panels) and 24 h (lower panels). The two panels on the right show enlarged images where the intracellular localization of fluorescent VLPs-cetuximab-Cy5.5 can be seen more clearly. Cell nuclei were dyed with DAPI (blue). Green areas represent the fluorescent VLPs either attached on the cell surface (cell membrane) or internalized (closer to the cell nucleus). One-micrometer-thick slices were made in the area equidistant between the apical and basal poles of the cells.

Article Snippet: On the other hand, the functionalization with the fluorescent dye took place by mixing 32 μg of purified VLP–Z domain (with and without previously linked cetuximab) with 3× molar excess of AmershamTM Cy5.5 mono NHS ester (GE Healthcare, Buckinghamshire, UK) to a final volume of 800 μL of HEPES 10 mM.

Techniques: Incubation, Membrane

Journal: bioRxiv

Article Title: SERF2, an RNA G-quadruplex Binding Protein, promotes stress granule formation

doi: 10.1101/2023.10.09.561572

Figure Lengend Snippet: (A) Phase regimes illustrating LLPS in SERF2 as a function of total RNA concentration extracted from HEK293T cells. (B) Fluorescence imaging shows gel-like structures in 50 µM SERF2 (left) mixed with 200 ng/µL of total RNA containing 10% (w/v) PEG8000 incubated for 30 minutes at room temperature. ( C-E ) 50 µM of SERF2 dissolved in 20 mM NaPi (pH 7.4), 100 mM KCl readily undergoes LLPS formation when mixed with equimolar concentration of rG4 quadruplexes that include TERRA, (G4C2) 4 , and (UG4U) 6 . The sample mixture contains 1/200 th Cy-5 label protein (purple signals) and 6-FAM rG4 quadruplex (green signals) as indicated in the figure inset. Two-component FRAP analysis are done to measure the protein recovery (purple plot) and rG4 quadruplex recovery (green plot) rates in the SERF2-rG4s droplets. The pre-bleached, after-bleached (0 s) and recovered droplets (300 s) are shown on the top of each FRAP plot. The FRAP data were fitted in GraphPad Prism using non-linear regression one-phase association model to obtain the recovery halftime (t1/2). (F,G) Schematics showing SERF2 and TERRA sample mixture phase separation in 10% PEG8000 at different protein to RNA concentrations (F), and at different salt and PEG8000 concentrations (G) . The black and purple spheres in all phase regime represents no formation or formation of droplets, respectively. (H) Dynamics and recovery of Cy5-labelled proteins in SERF2-total RNA droplets obtained by FRAP analysis suggest the gel-like structures are dynamic and reversible. Standard errors are calculated by analyzing 8 isolated droplets subjected to FRAP. (I) DIC and Fluorescence images showing co-phase separation of SERF2 (purple) and G3BP1 with HeLa total RNA. (J) SERF2 facilitates G3BP1-RNA condensation. 0, 25 or 50 µM of SERF2 protein was added to 12.5 ng/µl of HeLa total RNA with or without 25 µM of G3BP1.

Article Snippet: Cy-5 labeling was done by incubating 200 μM of SERF2 (T2C) with a 10-molar excess of Cy5 maleimide mono-reactive dye (Cytiva, PA25031) in 20 mM Tris-HCl pH7.4 and 100 mM KCl buffer overnight, at 25 °C, under continuous shaking at 300 rpm.

Techniques: Concentration Assay, Fluorescence, Imaging, Incubation, Isolation

Journal: PLoS ONE

Article Title: Non-Invasive In Vivo Imaging of Tumor-Associated CD133/Prominin

doi: 10.1371/journal.pone.0015605

Figure Lengend Snippet: (A) U251 s.c. xenograft mice [U251 wild-type cells (lower panel) and CD133-overexpressing U251 cells (upper panel)] were injected with 75 µg AC133.1-Cy5.5 and 2 nmol of IntegriSense 750. One, 2, 4 and 7 days later, the mice were imaged using the VisEn FMT-1500 Fluorescence Molecular Tomography system. The pictures presented correspond to the last measurement at day 7. (B) Quantification of AC133.1-Cy5.5 accumulation in U251 wild-type and CD133-overexpressing U251 xenografts. Signal intensity (pmol/mm 3 ) of the xenografts 7 days after AC133.1-Cy5.5 antibody injection (upper panel), and the resulting time course over 7 days (lower panel). (C) Signal intensity of isolated tumors 9 days after AC133.1-Cy5.5 injection. Tumors were resected from mice and imaged with an FMT-1500. WT, wild-type; OE, overexpressing.

Article Snippet: For large scale labeling, 1 mg purified AC133 antibody was incubated with 60 µM CyDye Cy5.5 mono-reactive NHS ester in 100 mM disodium hydrogen phosphate (pH 9) for 2 h. Labeled antibodies were purified using a PD10 column (GE Healthcare).

Techniques: Injection, Fluorescence, Tomography, Isolation

Journal: PLoS ONE

Article Title: Non-Invasive In Vivo Imaging of Tumor-Associated CD133/Prominin

doi: 10.1371/journal.pone.0015605

Figure Lengend Snippet: (A) HCT116 s.c. xenograft mice [HCT116 wild-type cells (upper panel) and HCT116 p53−/− cells (lower panel)] were injected with 75 µg AC133-Cy5.5 and 2 nmol of IntegriSense 750. One, 2, 3, 4 and 7 days later, the mice were imaged using the FMT-1500 system. The pictures presented correspond to the last measurement at day 7. (B) Quantification of AC133.1-Cy5.5 accumulation in HCT116 and HCT116 p53−/− xenografts. Comparison of signal intensities (pmol/mm 3 ) between the xenografts at day 7 after AC133.1-Cy5.5 injection (upper panel) and the time course over 7 days (lower panel). (C) Signal intensity of isolated tumors 9 days after AC133.1-Cy5.5 injection. Tumors were resected from mice and imaged with an FMT-1500 system. WT, wild-type.

Article Snippet: For large scale labeling, 1 mg purified AC133 antibody was incubated with 60 µM CyDye Cy5.5 mono-reactive NHS ester in 100 mM disodium hydrogen phosphate (pH 9) for 2 h. Labeled antibodies were purified using a PD10 column (GE Healthcare).

Techniques: Injection, Isolation

Journal: PLoS ONE

Article Title: Enhanced Nasal Mucosal Delivery and Immunogenicity of Anti-Caries DNA Vaccine through Incorporation of Anionic Liposomes in Chitosan/DNA Complexes

doi: 10.1371/journal.pone.0071953

Figure Lengend Snippet: Physicochemical characteristics of CS/DNA and AL/CS/DNA.

Article Snippet: Cy5.5 Mono NHS Ester was purchased from GE Healthcare (Buckinghamshire, UK).

Techniques:

Journal: PLoS ONE

Article Title: Enhanced Nasal Mucosal Delivery and Immunogenicity of Anti-Caries DNA Vaccine through Incorporation of Anionic Liposomes in Chitosan/DNA Complexes

doi: 10.1371/journal.pone.0071953

Figure Lengend Snippet: (A) Fluorescence images of nasal clearance of Cy5.5-CS/DNA and AL/Cy5.5-CS/DNA after i.n. administration. (B) Clearance derived from spectra. Fluorescence at t = 0 was set as 100%. (C) Adsorption of mucin on the CS/DNA and AL/CS/DNA. Formulations were prepared at pH 6.4; ** p <0.01 Mean ± SD (n = 4).

Article Snippet: Cy5.5 Mono NHS Ester was purchased from GE Healthcare (Buckinghamshire, UK).

Techniques: Fluorescence, Derivative Assay, Adsorption