Application suite x

The mitochondrial phenotypic changes in cells infected with SARS-CoV-2 virus was evaluated using genetically encoded mitochondrial sensors and mitochondria staining dyes. hiPSC derived cardiomyocytes were grown on 25-mm gelatin-coated glass coverslips and were infected with Ad-mito-GCamp6 (ex/em 480/510 nm: MOI 5). After 48 h, the cells were infected with SARS-CoV-2 virus (MOI 1) for 24 h and fixed with 10% Neutral buffered formalin. The fixed cells were imaged on a Leica TCS SP8 confocal imaging system using a 100x oil objective. The levels of mitophagy and mitochondrial phenotype was imaged, and the mitochondrial length, area and perimeter were measured using the Leica Application Suite X. The results were tabulated and plotted using GraphPad Prism version 8.
hiPSC derived cardiomyocytes were grown on 25-mm gelatin-coated glass coverslips and were infected with SARS-CoV-2 virus (MOI 1) for 24 h and were stained with Mitotracker deep red FM (ex/em 644/665 nm) for 30 min. The stained cells were fixed with 10% Neutral buffered formalin. The fixed cells were imaged on a Leica TCS SP8 confocal imaging system using a 100x oil objective. The levels of mitophagy mitochondrial phenotype i.e., mitochondrial length, area and perimeter were measured using the Leica Application Suite X. The results were tabulated and plotted using GraphPad Prism version 8.

Pupae were imaged whole with a Leica Fluorescence Stereo Microscope and Leica Application Suite X (LasX, Version 3.5.2.18963).
Confocal fluorescent images were taken with a Leica TCS SP8 with an HC PL APO CS2 63×/1.4 oil objective and Leica Application Suite X (LasX, Version 3.5.2.18963). Live imaging of macrophage cultures was performed using a Zeiss CellObserver Z.1 with a Yokogawa CSU-X1 spinning disk scanning unit and an Axiocam MRm CCD camera (6.45 µm×6.45 µm) and ZenBlue 2.5 software. Ablation experiments were performed using the UV ablation system DL-355/14 from Rapp OptoElectronics, as reported previously (Lehne et al., 2022 (link)).

hiPSC derived cardiomyocytes or COS-7 cells transiently transfected with the mRFP-tagged SARS-CoV-2 plasmid constructs were grown on 25-mm 0.1% gelatin or collagen coated glass coverslips, respectively. 48 h post-transfection, cells were loaded with 1 μM of ER Tracker blue-white DPX (ex/em 374/430 nm) and mitochondrial marker Dihydrorhodamine (2.5 μM; ex/em 505/524 nm) in cell growth media for 30 min at 37°C (5% CO2). The cells were washed and imaged within a temperature-controlled environmental chamber set at 37°C using a Leica TCS SP8 live-cell confocal imaging system. The subcellular localization of the cells transfected with SARS-CoV-2 proteins was imaged using a 100x oil objective and analyzed using Leica Application Suite X. The colocalization of Cox-8a mRFP and the SARS-CoV-2 proteins (Vector control, M Protein, Orf9c, Orf3a, Nsp6, Nsp10) with the ER (ER BFP) and mitochondria (DHR 123) were quantitatively measured using the Leica Application Suite X. The levels of colocalization were plotted by calculating the Pearson's co-efficient of colocalization and overlap co-efficient and the % of colocalization was determined. The values were plotted using GraphPad Prism version 8.

To confirm the correlation between the virus, Rap1b, and/or the cytoskeleton, or their relationship after treatments with chemicals or plasmids in HSV-1-infected cells, target-protein-specific primary antibodies, fluorescent-labeled secondary antibodies, or a fluorescently labeled chemical (phalloidin) were applied in IFA as previously described [26 (link),31 (link)]. Cell climbing sheets and cell plates were observed with a Leica SP8 confocal system (Leica Microsystems, Wetzlar, Germany). All images were captured and processed using Leica Application Suite X (Leica Microsystems).

PEDV-infected or non-infected Vero cells and BM-DCs in cell culture plates were fixed with 4% paraformaldehyde (Sigma-Aldrich), permeabilized with PBS containing 0.5% Triton X-100 (Sigma-Aldrich), then blocked with PBS containing 1% BSA (Sigma-Aldrich). Next, the cells were stained with mouse serum against PEDV-N protein. Specific binding between antibodies and corresponding targets was detected using Alexa Fluor^® 555-labeled goat anti-mouse IgG (Thermo Fisher Scientific). Cellular nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) at 37°C for 10 min and observed under a Leica DM1000 fluorescence microscope (Leica Microsystems, Wetzlar, Germany). All images were captured and processed using Leica Application Suite X (Leica Microsystems).