Journal: Microbiology Spectrum

Article Title: Bi-Reporter Vaccinia Virus for Tracking Viral Infections In Vitro and In Vivo

doi: 10.1128/Spectrum.01601-21

Figure Lengend Snippet: Direct visualization of viral infection in lungs of BALB/c mice infected with bi-reporter expressing rVV: Six-to-8-weeks-old female BALB/c mice ( n = 4) were mock-infected or infected with 10 7 PFU of WR, rVV Nluc/Scarlet, or rVV Nluc/GFP. Mice were sacrificed on days 2 and 4 postinfection and lungs were harvested to assess fluorescent Scarlet and GFP expression. Representative images from lungs of mock-infected and mice infected with WR, rVV Nluc/GFP, or rVV Nluc/Scarlet viruses on days 2 and 4 without immunostaining (A), stained with antibodies against Scarlet or GFP (B) or a rabbit polyclonal antibody against VV (C) are shown. Airways are noted with an asterisk. Scale bars 100 μm. Morphometric analyses of the infected bronchi in these experiments are also shown (D). One-way ANOVA was used for statistical analysis; ***, P < 0.001; n.s., no significance. The line represents the geometric mean.

Article Snippet: Primary antibodies include anti-NLuc rabbit polyclonal (1:6,000) (Promega), anti-mCherry rabbit polyclonal (1:5,000) (kindly provided by Dr. Antonio Alcamí), anti-GFP rabbit polyclonal (1:1,000) (Chemicon) rat monoclonal antibody 15B6, specific for F13 (1:50) (kindly provided by Dr. Gerhard Hiller), and anti-Actin monoclonal (1:500) (Sigma).

Techniques: Infection, Expressing, Immunostaining, Staining

Journal: Scientific Reports

Article Title: In vivo imaging of long-term accumulation of cancer-derived exosomes using a BRET-based reporter

doi: 10.1038/s41598-020-73580-5

Figure Lengend Snippet: PC3/CD63-Antares2 xenograft mice model enables continuous exosome tracking analysis in vivo. ( a ) Timeline of in vivo exosome tracking analysis in a xenograft mouse model established using PC3/CD63-Antares2- and EGFP-expressing (PC3/CD63-Antares2/EGFP) cells. Whole body imaging analyses using IVIS and BRET intensity measurement in blood samples were performed every 5 days after subcutaneous inoculation of control (PC3/Mock) or PC3/CD63-Antares2/EGFP cells into the mice. ( b ) Tumor growth in control (PC3/Mock) and PC3/CD63-Antares2/EGFP xenograft mice. ( c ) Time course of BRET signal in the blood of control (PC3/Mock) and PC3/CD63-Antares2/EGFP xenograft mice. ( d ) Sequential bioluminescence images of the whole body of control (PC3/Mock) and PC3/CD63-Antares2/EGFP xenograft mice. BRET emission derived from Antares2 was imaged using IVIS. ( e ) Bioluminescence images of organs harvested from control (PC3/Mock) and PC3/CD63-Antares2/EGFP tumor-bearing mice 5 weeks after inoculation. BRET emission derived from Antares2 and EGFP fluorescence were imaged using IVIS. ( f ) Immunohistochemical analysis of Nluc (Antares2) in the lungs, spleen, mesenteric nodes, and adipose tissues from mice bearing PC3/CD63-Antares2/EGFP cells (40 ×). Boxed with dotted line images are enlarged inset of the panels. Scale bar = 100 μm. Representative images from three mice are shown in (d, e, and f). Means ± SDs of tumor volume and luminescence intensity of BRET were obtained from three mice (b and c).

Article Snippet: Anti-Nluc rabbit polyclonal antibody was kindly provided by Promega (Madison, WI, USA).

Techniques: In Vivo, Expressing, Imaging, Control, Derivative Assay, Fluorescence, Immunohistochemical staining

Journal: Scientific Reports

Article Title: In vivo imaging of long-term accumulation of cancer-derived exosomes using a BRET-based reporter

doi: 10.1038/s41598-020-73580-5

Figure Lengend Snippet: The PC3/CD63-Antares2 xenograft mouse model is useful for evaluating the effects of exosome inhibitors in vivo. ( a ) Numbers of exosomes produced by PC3/CD63-Antares2 cells treated with DMSO or dasatinib (50 or 100 nM). ( b ) Timeline of in vivo exosome tracking analysis using the PC3/CD63-Antares2 xenograft mouse model. Dasatinib was administrated intraperitoneally once every 3 days. Whole body imaging using IVIS and BRET intensity measurement using blood samples were performed every 5 days after subcutaneous inoculation of PC3/CD63-Antares2 cells into the mice. ( c ) PC3/CD63-Antares2 cells were inoculated subcutaneously into the mice. When the tumor volume reached approximately 100 mm 3 , saline (Control) or dasatinib (2.5 mg/kg or 5 mg/kg) was administrated. Mean ± SD values of tumor volume (mm 3 ) obtained from 3 mice are plotted against days after inoculation. ( d ) Sequential BRET signal in the blood samples from control (saline administrated) or dasatinib-administrated (2.5 mg or 5 mg/kg) PC3/CD63-Antares2 xenograft mice. Means ± SDs of BRET signal obtained from three mice are plotted against days after inoculation. ( e ) Bioluminescence images of organs excised from control (saline-administrated) and dasatinib-administrated PC3/CD63-Antares2 xenograft mice 5 weeks after inoculation. (f) Immunohistochemical analysis of Nluc (Antares2) in the lungs and spleen from control (saline administrated) and dasatinib-administrated PC3/CD63-Antares2 xenograft mice (40 ×). Boxed in dotted line images are enlarged inset of the panels. Scale bar = 100 μm. * P < 0.05, ** P < 0.01, by ANOVA with Dunnett's post hoc analysis.

Article Snippet: Anti-Nluc rabbit polyclonal antibody was kindly provided by Promega (Madison, WI, USA).

Techniques: In Vivo, Produced, Imaging, Saline, Control, Immunohistochemical staining