Journal: The Journal of General Virology

Article Title: Interferon-β and mycophenolic acid are potent inhibitors of Middle East respiratory syndrome coronavirus in cell-based assays

doi: 10.1099/vir.0.061911-0

Figure Lengend Snippet: High content imaging of the MPA, ribavirin, IFN-α2a and IFN-β dose–response in MERS-CoV-infected Vero E6 cells. Vero E6 cells were treated with threefold dilutions of MPA and ribavirin (0.75–75 µM), and threefold dilutions of IFN-α2a and IFN-β (5–500 U ml−1), and subsequently infected at an m.o.i. of 0.1 with MERS-CoV. (a) At 48 h post-inoculation, cells were fixed and stained with antibody to MERS-CoV S protein and Alexa Fluor 594-conjugated secondary antibody. High content imaging analysis (Operetta, Harmony 3.1) was performed to determine the percentage of infected cells per well, and Hoechst 33342 staining was used to determine the number of viable cells in each well. One out of nine acquired fields per well is shown. The left-hand column shows the positive control of cells infected with MERS-CoV without drug treatment. The right-hand column shows the negative control of uninfected viable cells without drug treatment. (b) Quantification of relative fluorescence intensity from fluorescence microscopic images shown in (a). Mean fluorescence intensity (MFI) was measured in relative fluorescence units (RFU) and normalized to the number of cells per well. Results are representative of one experiment (mean±sd, n = 4). The experiment was repeated at least twice.

Article Snippet: The Operetta high content imaging system (PerkinElmer) and analysis software (Harmony 3.1) was used to quantify fluorescence of both dyes, Alexa Fluor 594 and Hoechst 33342.

Techniques: Imaging, Infection, Staining, Positive Control, Negative Control, Fluorescence

Journal: Respiratory Research

Article Title: GlyPerA™ effectively shields airway epithelia from SARS-CoV-2 infection and inflammatory events

doi: 10.1186/s12931-023-02397-3

Figure Lengend Snippet: GlyPerA™ shields primary HAE cells from SARS-CoV-2 Omicron BA.1 and BA.2 infection and innate immune activation. Visualization of virus binding (SARS-CoV-2 S1/N, orange) and complement (C3-FITC, green) in SARS-CoV-2 infected 3D pseudostratified epithelia. Pseudostratified epithelia were apically treated with GlyPerA™ prior exposure to SARS-CoV-2. On 3 dpi, filters were fixed, stained for höchst (blue), SARS-CoV-2 S1/N (orange), complement C3 (green) and acetylated tubulin (red) and then analysed by HCS. a XYZ-stacks of uninfected (UI, panel 1), BA.2-infected (panel 2), GlyPerA™-pre-treated and BA.2-infected (panel 3) HAE cultures were analyzed using the Operetta CLS HCS and the 63XWATER objective. Cells were stained using C3-FITC (green) as indicator for innate immune activation, SARS-CoV-2-S1/N-Alexa594 (orange) for virus detection, höchst for imaging nuclei (blue) and acetylated tubulin for staining cilia (red). High IC C3 mobilization was monitored in BA.2-infected cultures, while no virus and low C3 signals were detected in UI (panel 1) and GlyPerA™/BA.2- (panel 3) cultures. Scale bars represent 50 µm (XY) and 20 µm (YZ, XZ), respectively. Three independent experiments were performed. b Numbers of nuclei per condition (UI, Omicron, GlyPerA dil. PRE Omicron, GlyPerA dil. SIM Omicron), viral signals and c areas of C3 production were absolutely quantified from at least three different areas using the Harmony™ 4.9 software. Statistical significances were analyzed with GraphPad Prism software using One-way ANOVA and Tukey’s post test. In b and c **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: To study these complex models using primary cell cultured in 3D and to generate detailed phenotypic fingerprints for deeper biological insights in a high throughput manner, the Operetta CLS System (PerkinElmer, Waltham, MA, USA) was applied.

Techniques: Infection, Activation Assay, Virus, Binding Assay, Staining, Imaging, Software