mdck epithelial cells atcc  (ATCC)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Enhanced visualization of influenza A virus entry into living cells using virus-view atomic force microscopy

doi: 10.1073/pnas.2500660122

Figure Lengend Snippet: Morphological imaging of the live MDCK cell surface. Time-lapse AFM imaging showing representative membrane morphological dynamics at 27°C at 10-s intervals (related to Fig. 1C and SI Appendix, Fig. S1A). The topographic AFM image displays height variations up to 400 nm, where brighter areas indicate higher regions and darker areas indicate lower regions. Image size: 6.0 × 4.5 μm 2 .

Article Snippet: MDCK cells (CRL-34, American Type Culture Collection, Manassas, VA) were cultured under a 5% CO 2 humidified atmosphere at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Enhanced visualization of influenza A virus entry into living cells using virus-view atomic force microscopy

doi: 10.1073/pnas.2500660122

Figure Lengend Snippet: IAV lateral diffusion on the live MDCK cell surface. Time-lapse ViViD-AFM imaging showing representative IAV-AF594 virions diffusing on the cell membrane (related to Fig. 1E–F and SI Appendix, Fig. S3). Sequential images were acquired at 10-s intervals at 27°C. The movie shows the morphology of the cell surface virion (left), IAV-AF594 fluorescence (middle), and the merged image of morphology and fluorescence (right). Image size: 6.0 × 4.5 μm 2 . AFM height range: 400 nm. Arrowheads indicate three virions on the cell membrane.

Article Snippet: MDCK cells (CRL-34, American Type Culture Collection, Manassas, VA) were cultured under a 5% CO 2 humidified atmosphere at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Enhanced visualization of influenza A virus entry into living cells using virus-view atomic force microscopy

doi: 10.1073/pnas.2500660122

Figure Lengend Snippet: IAV cell surface diffusion on live MDCK cell. Time-lapse AFM imaging showing representative IAV virion diffusion on the cell membrane from t=225 s to 575 s (related to Fig. 2A–C). Sequential images were acquired at 5-s intervals at 37°C. Arrowhead indicates the tracked virion. AFM image: 6.0 × 4.5 μm 2 . AFM height range: 400 nm.

Article Snippet: MDCK cells (CRL-34, American Type Culture Collection, Manassas, VA) were cultured under a 5% CO 2 humidified atmosphere at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Enhanced visualization of influenza A virus entry into living cells using virus-view atomic force microscopy

doi: 10.1073/pnas.2500660122

Figure Lengend Snippet: IAV cell surface diffusion on live MDCK cell before and after clathrin assembly. Time-lapse ViViD-AFM imaging (t=0 s to 80 s) showing IAV virion diffusion on the cell membrane before and after the onset of clathrin assembly at t=50 s (related to Fig. 3A–D). Sequential images were acquired at 5-s intervals at 37°C, showing: morphology of virion and cell membrane (left), IAV-AF594 fluorescence (magenta, middle), and CLCa-EGFP fluorescence (green, right). Arrowheads indicate the virion position in AFM images, IAV-AF594 fluorescence, and the site of clathrin assembly. Image size: 6.0 × 4.5 μm 2 . AFM height range: 400 nm. Clathrin assembly at the arrowhead position is observed from t=50 s.

Article Snippet: MDCK cells (CRL-34, American Type Culture Collection, Manassas, VA) were cultured under a 5% CO 2 humidified atmosphere at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Enhanced visualization of influenza A virus entry into living cells using virus-view atomic force microscopy

doi: 10.1073/pnas.2500660122

Figure Lengend Snippet: Moment of IAV internalization into an MDCK cell. Time-lapse AFM three-dimensional movie showing a representative IAV virion internalization event (related to Fig. 4A and B). Sequential images were acquired at 5-s intervals at 37°C. The virion was colored in magenta.

Article Snippet: MDCK cells (CRL-34, American Type Culture Collection, Manassas, VA) were cultured under a 5% CO 2 humidified atmosphere at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Enhanced visualization of influenza A virus entry into living cells using virus-view atomic force microscopy

doi: 10.1073/pnas.2500660122

Figure Lengend Snippet: Tracking of an IAV virion on an MDCK cell before and after CME. Time-lapse ViViD-AFM imaging showing the virion trajectory from t=-230 s to 180 s, with internalization occurring at t=0 s (related to SI Appendix, Fig. S8). Sequential images were acquired at 5-s intervals at 37°C, showing the merged fluorescence of CLCa -EGFP (green) and IAV-AF594 (magenta) (left), AFM morphology of virion and cell membrane (middle) and merged fluorescence and morphological data (right). Arrowhead indicates the virion morphology visible by AFM. In fluorescence images, the tracked virion trajectory is overlaid (orange: extracellular, gray: intracellular). Image size: 6.0 × 8.6 μm 2 (fluorescence, left) and 6.0 × 4.5 μm 2 (AFM and merged images, middle and right). AFM height range: 400 nm.

Article Snippet: MDCK cells (CRL-34, American Type Culture Collection, Manassas, VA) were cultured under a 5% CO 2 humidified atmosphere at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Enhanced visualization of influenza A virus entry into living cells using virus-view atomic force microscopy

doi: 10.1073/pnas.2500660122

Figure Lengend Snippet: Effect of EIPA treatment on the live MDCK cell surface morphology (related to SI Appendix, Fig. S12A). Time-lapse AFM imaging showing dynamics of the MDCK cell surface morphology before and after 20-min EIPA treatment at 37°C. Sequential images were acquired at 5-s intervals at 37°C. Image size: 6.0 × 4.5 μm 2 . AFM height range: 400 nm.

Article Snippet: MDCK cells (CRL-34, American Type Culture Collection, Manassas, VA) were cultured under a 5% CO 2 humidified atmosphere at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Enhanced visualization of influenza A virus entry into living cells using virus-view atomic force microscopy

doi: 10.1073/pnas.2500660122

Figure Lengend Snippet: IAV CME in an EIPA-treated MDCK cell (related to SI Appendix, Fig. S12B). Time-lapse AFM imaging showing representative membrane morphological changes during IAV CME, with internalization occurring at t=0 s. Sequential images were acquired at 5-s intervals at 37°C. The virion was colored in magenta. Scale bar: 1000 nm. AFM height range: 400 nm.

Article Snippet: MDCK cells (CRL-34, American Type Culture Collection, Manassas, VA) were cultured under a 5% CO 2 humidified atmosphere at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Enhanced visualization of influenza A virus entry into living cells using virus-view atomic force microscopy

doi: 10.1073/pnas.2500660122

Figure Lengend Snippet: IAV cell surface diffusion before and after clathrin assembly. MDCK cells expressing clathrin light chain a (CLCa) fused with EGFP (CLCa-EGFP) were inoculated with IAV/WSN-AF594 at 37 °C and imaged using ViViD-AFM at 5-s intervals. ( A ) Time-lapse detection of a diffusing IAV/WSN virion (yellow and orange arrowheads) and clathrin assembly (white arrowheads). Image size: 2,000 × 2,000 nm 2 . AFM height range: 400 nm. ( B − D ) Analysis of cell surface IAV diffusion for the virion shown in panel ( A ). ( B ) CLCa fluorescence (FL) intensity at virion attachment sites over time. Clathrin assembly onset was defined as the time when CLCa FL intensity exceeded the background level (BG) indicated by a dashed line. AU, arbitrary units. ( C ) Trajectory of IAV/WSN-AF594 virion on the cell surface, color-coded to show movement before (pink) and after (brown) onset of clathrin assembly. (Scale bar, 500 nm.) ( D ) Analysis of single virion diffusion coefficient before and after onset of clathrin assembly. Diffusion coefficient ( D ) is indicated on the graph. MSD, mean squared displacement. ( C and D ) The trajectory of IAV and the analysis of diffusion coefficient were obtained based on fluorescence data. ( E ) Diffusion coefficients of individual virions before and after clathrin assembly under control (N = 7), sialidase-treated (N = 3), and oseltamivir-treated (N = 3) conditions, compared with virus-free CCPs (mean = −3.76, N = 33). Dashed lines connect the measurements of individual virions.

Article Snippet: MDCK cells (CRL-34, American Type Culture Collection, Manassas, VA) were cultured under a 5% CO 2 humidified atmosphere at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific).

Techniques: Diffusion-based Assay, Expressing, Fluorescence, Control, Virus

Journal: Heliyon

Article Title: Rapid in-EPON CLEM: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes and StayGold

doi: 10.1016/j.heliyon.2024.e28055

Figure Lengend Snippet: Workflow of sample preparation for in-resin CLEM. HeLa cells expressing self-labeling enzyme (SLE) fusions were stained with ligand-conjugated dyes before first fixation with glutaraldehyde (GA) and paraformaldehyde (PFA). If indicated, cells were experimentally treated, e. g. infected, transfected, or pulse/chased with fluid tracers, Cells of interest were selected by light microscopy (LM). Subsequently, samples were prepared for electron microscopy (EM) by further fixation with GA, post-fixation with osmium tetroxide, dehydration in ethanol, and embedding in EPON. Sections were generated and registered by widefield LM, followed by contrasting and imaging by transmission electron microscopy (TEM). Finally, the LM and EM modalities were overlaid and correlated. *Steps marked by asterisks were omitted for quantitative comparison of various dyes.

Article Snippet: The epithelial cell line HeLa (American Type Culture Collection, ATCC no. CCL-2) and derivatives ( ) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 4.5 g/l glucose, 4 mM stable glutamine and sodium pyruvate (Biochrom), and 10% inactivated fetal calf serum (iFCS) (Sigma-Aldrich) at 37 °C in an atmosphere containing 5% CO 2 and 90% humidity.

Techniques: Sample Prep, Expressing, Labeling, Staining, Infection, Transfection, Light Microscopy, Electron Microscopy, Generated, Imaging, Transmission Assay, Comparison

Journal: Heliyon

Article Title: Rapid in-EPON CLEM: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes and StayGold

doi: 10.1016/j.heliyon.2024.e28055

Figure Lengend Snippet: The red fluorescent dyes JF549, JFX549, JFX554, and TMR retain fluorescence after CE in EPON. HeLa cells stably expressing Tom20-HaloTag were stained with the indicated HTL conjugates with fluorescence emission in green (A), red (B), or far-red (C) spectra at the indicated concentrations for 30 min. Cells were fixed with glutaraldehyde and osmium tetroxide, and embedded in EPON resin at 60 °C for 48 h. Next, the samples were sectioned into 250 nm sections, and placed on a glass coverslip for imaging by widefield microscopy using an Olympus LSM FV3000 NLO. Analysis of the acquired images revealed loss of fluorescence for the green JF479 at both concentrations, for the fluorogenic red dye JF585, as well as for all tested far-red dyes, while fluorescence signals being reminiscent of mitochondria were detected for JF549, JFX549, JFX554, and TMR. The far-red dyes showed homogenous cellular fluorescence at dye concentration of 1 μM. D) Control HeLa Tom20-HaloTag cells without labeling with HTL conjugates were imaged in the green, red, and far-red channel and show background fluorescence of EPON, especially in the green channel. E) Quantification of signal intensities. Line scans of 50 μm (yellow lines in micrographs) were quantified for gray values expressed as arbitrary units (a. u.) over distance. Peaks in the plots represent either a specific fluorescence or parts of the speckled background resulting from EPON embedding (see e. g. peak from JF646). Scale bars: 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The epithelial cell line HeLa (American Type Culture Collection, ATCC no. CCL-2) and derivatives ( ) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 4.5 g/l glucose, 4 mM stable glutamine and sodium pyruvate (Biochrom), and 10% inactivated fetal calf serum (iFCS) (Sigma-Aldrich) at 37 °C in an atmosphere containing 5% CO 2 and 90% humidity.

Techniques: Fluorescence, Stable Transfection, Expressing, Staining, Imaging, Microscopy, Concentration Assay, Control, Labeling

Journal: Heliyon

Article Title: Rapid in-EPON CLEM: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes and StayGold

doi: 10.1016/j.heliyon.2024.e28055

Figure Lengend Snippet: Comparison of fluorescence intensities and signal-to-background ratios of TMR, JF549, JFX549, and JFX554 in resin. HeLa cells stably expressing Tom20-HaloTag were stained with 100 nM of indicated HTL dye for 30 min. Cells were fixed and prepared as in . The average signal intensity and standard deviations were ascertained by the algorithm for the selection and the background of each acquired image. A) Mean signal intensities of all evaluated images. The standard deviation was computed by averaging the standard deviation determined by the algorithm for the signal intensity of the selection and is indicated by the whiskers. ii) displays the same as i) but subdivided into the individual experiments. B) Signal-to-background ratios (S/B) were computed by dividing the average signal intensity of the selection by the average signal intensity of the background for each image. Results of individual experiments were shown in the right panel, and indicate that labeling with HTL conjugated to TMR or JF549 frequently failed, while conjugates with JFX549 or JFX554 labeled in all cases. Only experiments resulting in detectable fluorescence were included in the calculation of means. Statistical analyses were performed by one-way ANOVA test and the Bonferroni t -test. Significances are indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The epithelial cell line HeLa (American Type Culture Collection, ATCC no. CCL-2) and derivatives ( ) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 4.5 g/l glucose, 4 mM stable glutamine and sodium pyruvate (Biochrom), and 10% inactivated fetal calf serum (iFCS) (Sigma-Aldrich) at 37 °C in an atmosphere containing 5% CO 2 and 90% humidity.

Techniques: Comparison, Fluorescence, Stable Transfection, Expressing, Staining, Selection, Standard Deviation, Labeling

Journal: Heliyon

Article Title: Rapid in-EPON CLEM: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes and StayGold

doi: 10.1016/j.heliyon.2024.e28055

Figure Lengend Snippet: In-resin CLEM and tomography on 250 nm semithin or 100 nm thin sections after room temperature fixation or high-pressure freezing. HeLa cells stably expressing Tom20-HaloTag were stained with 100 nM HTL-JFX554 for 30 min and processed for in-resin CLEM via CE preparation (A–D) or HPF and FS (E, F). Sections of 250 nm thickness ( A, B, E, F ) were prepared for TEM tomography ( Bi, see for tomogram; Fi, see for tomogram), and 100 nm sections ( C, D ) were imaged using a standard TEM setup ( Ci, Di ). In all cases, fluorescence was retained after sample preparation and allowed precise correlation of labeled mitochondria within the TEM images ( Aii, Bii, Cii, Dii, Eii, Fii ). Scale bars: A, Ai, Aii, C, Ci, Cii: 10 μm; E, Ei, Eii: 5 μm; B, Bi, Bii, D, Di, Dii, F, Fi, Fii: 1 μm.

Article Snippet: The epithelial cell line HeLa (American Type Culture Collection, ATCC no. CCL-2) and derivatives ( ) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 4.5 g/l glucose, 4 mM stable glutamine and sodium pyruvate (Biochrom), and 10% inactivated fetal calf serum (iFCS) (Sigma-Aldrich) at 37 °C in an atmosphere containing 5% CO 2 and 90% humidity.

Techniques: Tomography, Stable Transfection, Expressing, Staining, Fluorescence, Sample Prep, Labeling

Journal: Heliyon

Article Title: Rapid in-EPON CLEM: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes and StayGold

doi: 10.1016/j.heliyon.2024.e28055

Figure Lengend Snippet: Various cellular structures can be addressed by in-resin CLEM. HeLa ( A , D , E , F ) or HEK ( B , C ) cells were transiently transfected for expression of Golgi-HaloTag ( A ), Connexin36-SNAP-tag ( B , C ), LAMP1-HaloTag ( D , E ), or the Salmonella Typhimurium effector protein SseG-HaloTag ( F ), stained with 100 nM HTL-JFX554 or HTL-TMR for 30 min, and conventionally prepared for EM. Sections of 250 nm ( A, B, C, D, E ) and 100 nm ( F ) thickness were prepared and imaged by LM ( A, B, C, D, E, F ). Fluorescence signals were retained allowing identification of labeled structures. Next, TEM tomography ( Ei ) or standard TEM imaging ( Ai, Bi, Ci, Di, Dii, Fi, Fii ) was performed, and precise correlations of the Golgi apparatus ( Ai, G ) or ER whorls ( Ci ) were obtained. HeLa cells expressing LAMP1-HaloTag were infected with S. Typhimurium, and 8 h post infection cells were stained with HTL-JFX554 and conventionally prepared for EM ( D, E, see for tomogram). Tubular LAMP1-positive structures induced by Salmonella are clearly visible. A branching SIF can be observed in LM and TEM tomography ( E, Ei, Eii ). Correlation reveals the tubular architecture of LAMP1-positive membranes ( Eii ). In an uninfected control cell, the endo-lysosomal system is not modified (Eiii). After expression in HeLa cells, SseG localizes at various compartments of the Golgi apparatus ( F, Fi, Fii ). Scale bars: A, Ai: 2 μm; B, Bi: 5 μm; C, Ci: 500 nm; D, Di, Dii, Eiii: 10 μm; E, Ei, Eii, F, Fi, Fii: 1 μm.

Article Snippet: The epithelial cell line HeLa (American Type Culture Collection, ATCC no. CCL-2) and derivatives ( ) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 4.5 g/l glucose, 4 mM stable glutamine and sodium pyruvate (Biochrom), and 10% inactivated fetal calf serum (iFCS) (Sigma-Aldrich) at 37 °C in an atmosphere containing 5% CO 2 and 90% humidity.

Techniques: Transfection, Expressing, Staining, Fluorescence, Labeling, Tomography, Imaging, Infection, Control, Modification

Journal: Heliyon

Article Title: Rapid in-EPON CLEM: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes and StayGold

doi: 10.1016/j.heliyon.2024.e28055

Figure Lengend Snippet: Application of HaloTag ligands with Janelia Fluor dyes for in-resin super-resolution microscopy and CLEM. HeLa cells stably expressing Tom20-HaloTag were labeled with 100 nM HTL-JFX554 for 30 min and conventionally prepared for EM. 250 nm semithin sections were prepared and subjected to Lattice Light-Sheet Microscopy (LLSM) in combination with Structured Illumination Microscopy (SIM, A, B, C ). The comparison of images obtained by LLSM ( B ) or SIM ( C ) modalities indicates the improved spatial resolution of SIM. After performing TEM tomography ( D, see for tomogram), the super-resolved LM image was precisely correlated with mitochondrial membranes ( E, M). LLSM-SIM was also possible on 100 nm thin sections of HeLa cells stably transfected with LAMP1-HaloTag and labeled with 100 nM JFX554 for 30 min ( F, Fi, Fii ). Scale bars: A: 5 μm; B, C, D, E: 2 μm; F, Fi, Fii: 10 μm.

Article Snippet: The epithelial cell line HeLa (American Type Culture Collection, ATCC no. CCL-2) and derivatives ( ) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 4.5 g/l glucose, 4 mM stable glutamine and sodium pyruvate (Biochrom), and 10% inactivated fetal calf serum (iFCS) (Sigma-Aldrich) at 37 °C in an atmosphere containing 5% CO 2 and 90% humidity.

Techniques: Super-Resolution Microscopy, Stable Transfection, Expressing, Labeling, Microscopy, Comparison, Tomography, Transfection

Journal: Heliyon

Article Title: Rapid in-EPON CLEM: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes and StayGold

doi: 10.1016/j.heliyon.2024.e28055

Figure Lengend Snippet: Dual-fluorescence in-resin CLEM using combinations of HaloTag and SNAP-tag, or HaloTag and fluorescent protein StayGold. HeLa cells stably expressing Tom20-HaloTag were transiently transfected for expression of Connexin36-SNAP-tag ( A, B) or ER-StayGold ( C, D ), labeled with 100 nM HTL-JFX554 (magenta) and SNAP-tag ligand-JF525 (yellow) for 30 min and conventionally prepared for EM. 250 nm semithin sections were prepared and subjected to dual-fluorescence LM ( A, Bi, C, Ci, Cii ). Fluorescence signals of JF525 and JFX554 were retained in resin, allowing simultaneous detection of ER whorls and mitochondria and their correlation ( A, Ai, Aii, B, Bi) . Fluorescence signals of StayGold (green) and JFX554 (magenta) were retained in resin, allowing correlation of ER and mitochondria within the tomographic volume towards detection of membrane contact sites ( C, D, Di, Dii, white arrowheads, ). Scale bars: A, Ai, Aii, B, Bi, D, Di, Dii: 1 μm; C: 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The epithelial cell line HeLa (American Type Culture Collection, ATCC no. CCL-2) and derivatives ( ) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 4.5 g/l glucose, 4 mM stable glutamine and sodium pyruvate (Biochrom), and 10% inactivated fetal calf serum (iFCS) (Sigma-Aldrich) at 37 °C in an atmosphere containing 5% CO 2 and 90% humidity.

Techniques: Fluorescence, Stable Transfection, Expressing, Transfection, Labeling, Membrane

Journal: Heliyon

Article Title: Rapid in-EPON CLEM: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes and StayGold

doi: 10.1016/j.heliyon.2024.e28055

Figure Lengend Snippet: Cell lines used in this study.

Article Snippet: The epithelial cell line HeLa (American Type Culture Collection, ATCC no. CCL-2) and derivatives ( ) were cultured in Dulbecco's modified Eagle medium (DMEM) containing 4.5 g/l glucose, 4 mM stable glutamine and sodium pyruvate (Biochrom), and 10% inactivated fetal calf serum (iFCS) (Sigma-Aldrich) at 37 °C in an atmosphere containing 5% CO 2 and 90% humidity.

Techniques: Stable Transfection, Transfection, Expressing

Journal: Pathogens

Article Title: Differing Expression and Potential Immunological Role of C-Type Lectin Receptors of Two Different Chicken Breeds against Low Pathogenic H9N2 Avian Influenza Virus

doi: 10.3390/pathogens13010095

Figure Lengend Snippet: H9N2 virus growth under overexpression or downregulation of CLR in DF-1 cells. Transfection of CLR expressing plasmids ( A ) or siRNA targeting CLR mRNA ( B ) was conducted 12 h before inoculating virus. Supernatant virus titers were measured at 12 h after transfection using MDCK cells in triplicate. The asterisk indicates statistical significance by one-way ANOVA and Tukey’s post hoc comparison ( p < 0.05).

Article Snippet: H9N2 virus was titrated in Madin-Darby canine kidney (MDCK) cells (ATCC, CRL-34).

Techniques: Virus, Over Expression, Transfection, Expressing, Comparison

Journal: Viruses

Article Title: Human Metapneumovirus-Induced Host microRNA Expression Impairs the Interferon Response in Macrophages and Epithelial Cells

doi: 10.3390/v15112272

Figure Lengend Snippet: Effect of hsa-miR-4634 on interferon expression in epithelial cells. ( A ) A549 cells were infected at MOI 0.5, 1.0, and 3.0, and the percentage of HMPV-positive cells and cell death was quantified by flow cytometry. ( B ) A549 cells were infected with HMPV at an MOI of 3.0 for 24 h, and the expression of hsa-miR-4634 was assessed by RT-qPCR. ( C – E ) A549 cells were transfected by electroporation with mimic-4634 (100 nM) or inhibitor-4634 (50 nM). After 24 h of culture, cells were infected with HMPV at an MOI of 3.0 for 24 h. Total RNA was extracted to assess the expression of IFN-α2, IFN-β, and IFN-λ2/3 by qPCR. Bar graphs represent mean of three independent experiments ± SEM. Statistical differences were calculated using the Student t -test ( B ) or ANOVA followed by Sidak’s multiple comparison test ( A , C – E ). * p < 0.05; ** p < 0.01, *** p < 0.001, **** p ≤ 0.0001.

Article Snippet: A549 cells, a human alveolar basal epithelial cell line derived from lung adenocarcinoma with type II alveolar epithelial cells (ATCC CCL-185C, Manassas, VA, USA), were cultivated in F12K medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% FBS (Gibco, Gaithersburg, MD, USA) and 1% penicillin-streptomycin (Gibco, Gaithersburg, MD, USA), herein referred as the complete medium.

Techniques: Expressing, Infection, Flow Cytometry, Quantitative RT-PCR, Transfection, Electroporation, Comparison

Journal: Viruses

Article Title: Human Metapneumovirus-Induced Host microRNA Expression Impairs the Interferon Response in Macrophages and Epithelial Cells

doi: 10.3390/v15112272

Figure Lengend Snippet: Effect of hsa-miR-4634 on viral replication in macrophages and epithelial cells. Viral copies of the HMPV N gene were quantified by RT-qPCR. Cells were transfected by electroporation with mimic (100 nM) and inhibitor (50 nM). ( A ) Macrophages and ( B ) epithelial cells were infected for 24 h with HMPV at MOI 1 or MOI 3, respectively. Bar graphs represent the mean of two independent experiments with similar results ± SEM. Statistical differences were calculated using the Student t -test. * p < 0.05, *** p <0.001.

Article Snippet: A549 cells, a human alveolar basal epithelial cell line derived from lung adenocarcinoma with type II alveolar epithelial cells (ATCC CCL-185C, Manassas, VA, USA), were cultivated in F12K medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% FBS (Gibco, Gaithersburg, MD, USA) and 1% penicillin-streptomycin (Gibco, Gaithersburg, MD, USA), herein referred as the complete medium.

Techniques: Quantitative RT-PCR, Transfection, Electroporation, Infection

Journal: Journal of the Formosan Medical Association = Taiwan yi zhi

Article Title: Microbiological and genomic investigations of invasive Salmonella enterica serovar Panama from a large outbreak in Taiwan.

doi: 10.1016/j.jfma.2021.07.002

Figure Lengend Snippet: Figure 2 Virulence examination. Assays of (A) invasion (at 1st hour), (B) penetration through MDCK cells, (C) intracellular survival and (D) cytotoxicity of iNTS to THP-1-derived macrophages were evaluated using S. Panama and other NTS serovars, including S. Typhimurium SL1344, S. Choleraesuis SC-B67, S. Panama SP3464, S. Panama SP4156, S. Enteritidis SE4298, S. Enteritidis SE4312, S. Enteritidis SE3546, S. Enteritidis SE3675 and S. Enteritidis SE3694. N: nonpathogenic E. coli RDEC-1 as a negative control; STM: S. Typhimurium; SC: S. Choleraesuis; SP: S. Panama; SE: S. Enteritidis; CFU: colony formation unit. *: P < 0.05; **: P < 0.005; ***: P < 0.001; ns: no significant difference.

Article Snippet: The human monocytic leukemia cell line THP-1 (ATCC TIB-202 ) and the canine renal epithelial cell line MDCK (ATCC CCL-34 ) were used in this study.

Techniques: Derivative Assay, Negative Control