Journal: Viruses

Article Title: PluriBAC: A Versatile Baculovirus-Based Modular System to Express Heterologous Genes in Different Biotechnological Platforms

doi: 10.3390/v15101984

Figure Lengend Snippet: Per os infection of Spodoptera frugiperda larvae with PluriBAC-derived recAcMNPV. ( A ) A representation of the pathway followed in PluriBAC scheme to construct the virus, and the final pGGL2Bac donor vector obtained to generate the recAcMNPV. CMV represents cytomegalovirus immediate-early gene 1 (CMV-IE1) promoter and SV40 refers to simian virus 40 polyadenylation signal (SV40 polyA). ( B ) PCR screening of the insert: CMV + dTomato + SV40 (1650 bp) and fluorescent infected High Five ™ cells. ( C ) Experimental protocol (created with BioRender.com , accessed on 14 September 2023) and epifluorescence microscopy of infected and uninfected Spodoptera frugiperda larvae with recAcMNPV.

Article Snippet: Recombinant protein expression was verified with epifluorescence microscopy Nikon Eclipse Ti-S (Nikon Instruments Inc. Melville, NY, USA) and photographed with Nikon DS-Ri2 camera.

Techniques: Infection, Derivative Assay, Construct, Virus, Plasmid Preparation, Epifluorescence Microscopy

Journal: Viruses

Article Title: PluriBAC: A Versatile Baculovirus-Based Modular System to Express Heterologous Genes in Different Biotechnological Platforms

doi: 10.3390/v15101984

Figure Lengend Snippet: CNS cells transduction with PluriBAC-derived recAcMNPV. ( A ) A representation of the pathway followed in PluriBAC workflow to generate the virus, where CMV stands for cytomegalovirus immediate-early gene 1 (CMV-IE1) promoter and SV40, for simian virus 40 polyA signal (SV40 polyA). ( B ) PCR screening of the expression cassette (1485 bp) and fluorescent High Five ™ cells infected with the recAcMNPV obtained. ( C ) Experimental protocol (created with BioRender.com , accessed on 14 September 2023) and epifluorescence microscopy of mice astrocytes and patient-derived GBM cells transduced with recombinant baculovirus. Citrine expression was observed 48 h after transduction. ( D ) Experimental protocol (created with BioRender.com , accessed on 14 September 2023) of the in vivo assay and citrine expression observation of mice normal astrocytes. C57Bl/6 mice were inoculated into the right striatum with BV (5 × 10 8 PFU). After 7 days, the expression of the reporter protein citrine (green) was evaluated with fluorescent microscopy. ( E ) Experimental protocol (created with BioRender.com , accessed on 14 September 2023) of the in vivo assay and citrine expression observation of mouse neoplastic astrocytes. C57Bl/6 mice were inoculated with mouse glioma neurospheres (NS) into the brain; 3 weeks later, tumors were injected with BV (5 × 10 8 PFU) and mice were euthanized after 7 days. The expression of the reporter protein citrine (green) in the tumor was evaluated with fluorescent microscopy. In all cases, DAPI was used for nuclear staining.

Article Snippet: Recombinant protein expression was verified with epifluorescence microscopy Nikon Eclipse Ti-S (Nikon Instruments Inc. Melville, NY, USA) and photographed with Nikon DS-Ri2 camera.

Techniques: Transduction, Derivative Assay, Virus, Expressing, Infection, Epifluorescence Microscopy, Recombinant, In Vivo, Microscopy, Injection, Staining

Journal: Viruses

Article Title: PluriBAC: A Versatile Baculovirus-Based Modular System to Express Heterologous Genes in Different Biotechnological Platforms

doi: 10.3390/v15101984

Figure Lengend Snippet: Characterization of the induction of pMDR1 with cisplatin using a PluriBAC-derived AcMNPV encoding dTomato fluorescent protein. ( A ) A representation of the pathway followed in PluriBAC workflow to generate the baculovirus. SV40: simian virus 40 polyA signal. ( B ) PCR screening of the inserts: pMDR1 (1393 bp), dTomato + SV40 (864 bp), and obtained fluorescent High Five™ cells infected with the recAcMNPV. ( C ) Experimental protocol (created with BioRender.com , accessed on 14 September 2023) and epifluorescence microscopy of transduced A549 cells treated with cisplatin at different times (for practical purposes, only two cisplatin concentrations are represented). Percentages of positive cells were calculated in the different cisplatin concentration assayed at 72 h post-transduction. A Student’s t test was performed between groups, * p < 0.001.

Article Snippet: Recombinant protein expression was verified with epifluorescence microscopy Nikon Eclipse Ti-S (Nikon Instruments Inc. Melville, NY, USA) and photographed with Nikon DS-Ri2 camera.

Techniques: Derivative Assay, Virus, Infection, Epifluorescence Microscopy, Concentration Assay, Transduction

Journal: Viruses

Article Title: PluriBAC: A Versatile Baculovirus-Based Modular System to Express Heterologous Genes in Different Biotechnological Platforms

doi: 10.3390/v15101984

Figure Lengend Snippet: Multiple insert PluriBAC-derived recBV constructed using Bac-to-Bac ® expression system. ( A ) A representation of the pathway followed in PluriBAC scheme to construct the virus, and the final pGGL1-Bac donor vector obtained to generate the recAcMNPV with Bac-toBac TM expression system. Tn7L and Tn7R: transposition sites, pSpect: Spectinomycin resistance promoter, Spect R: Spectinomycin resistance, pSfU6: small nucleolar U6 Spodoptera frugiperda promoter, ncRNA: synthetic non-coding RNA, CMV: cytomegalovirus immediate-early gene 1 (CMV-IE1) promoter, SV40: simian virus 40 polyA signal. ( B ) PCR screening of the multiple inserts. Insert 1: Tn7R (260bp), insert 3: pSfU6 + ncRNA + term (520 bp), insert 4: CMV (507 bp), insert 5: Tn7L (211 bp). Spectinomycin resistance and its promoter were screened by using Spectinomycin for clonal selection. ( C ) Experimental protocol (created with BioRender.com , accessed on 14 September 2023) of recAcMNPV generation using Bac-to-Bac TM system. Blue–white transposition screening in E. coli DH 10Bac TM cells and epifluorescence microscopy of infected High Five TM cells with recBV. Yellow arrows show some of the white colonies obtained.

Article Snippet: Recombinant protein expression was verified with epifluorescence microscopy Nikon Eclipse Ti-S (Nikon Instruments Inc. Melville, NY, USA) and photographed with Nikon DS-Ri2 camera.

Techniques: Derivative Assay, Construct, Expressing, Virus, Plasmid Preparation, Single Photon Emission Computed Tomography, Selection, Epifluorescence Microscopy, Infection

Journal: Scientific Reports

Article Title: Epifluorescence-based three-dimensional traction force microscopy

doi: 10.1038/s41598-020-72931-6

Figure Lengend Snippet: The procedure for calculating tractions from epifluorescence imaging. Volumetric epifluorescent images of spherical fiducial markers (i.e., fluorescent microbeads) embedded at the surface of a cellular substrate medium are imported into Matlab where they are deconvolved and a particle tracking algorithm is employed to calculate 3D displacements. The nearly-planar scattered displacements are mapped onto a planar grid with a second order lookup-table regularization. These 3D surface displacements are then input into a finite-element based solution to the boundary value problem defined by the known and measured displacement fields and material properties. From this computation, the stresses and a full-field 3D displacement field in the substrate domain are extracted and used to compute in-plane and out-of-plane traction components. The tractions may then be used to compute biologically-relevant quantities of interest. The right-hand column provides a simplified illustration of the process for images taken at time, t , and after some time, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau$$\end{document} τ .

Article Snippet: Polyacrylamide gels with a single layer of fluorescent beads were imaged on a Nikon TI-2 epifluorescence microscope using a 40 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × /0.6 NA air objective with an aligned correction collar.

Techniques: Imaging

Journal: Scientific Reports

Article Title: Epifluorescence-based three-dimensional traction force microscopy

doi: 10.1038/s41598-020-72931-6

Figure Lengend Snippet: Typical single-layer beaded gel images from epifluorescence microscopy and resultant displacement reconstruction accuracy metrics. ( a ) Maximum intensity projections from both pre- and post-deconvolution results for an experimental image using the experimentally-gathered point spread function shown in the center column. The deconvolved point spread function is shown in the column on the right. Insets show a closer view of the maximum intensity z -projections of experimental images pre- and post-deconvolution. ( b ) Histograms showing the distributions of the reconstructed planarized displacement in x , y and z from a nominally zero-displacement experimental image pair and resultant x , y and z tractions. Note the zero-centered symmetric nature, indicating little bias or skew at the noise floor. ( c ) Line plot showing the measured mean displacement magnitude versus the applied experimental displacement magnitude from three individual rigid body displacement experiments, each imposing rigid motion in one of the three principal directions with sequentially increasing displacements along the indicated axis. Standard deviation is plotted as a shaded error bar (very small); inset shows the shaded error bars in more detail.

Article Snippet: Polyacrylamide gels with a single layer of fluorescent beads were imaged on a Nikon TI-2 epifluorescence microscope using a 40 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × /0.6 NA air objective with an aligned correction collar.

Techniques: Epifluorescence Microscopy, Standard Deviation