Axiovert 200 m system

Uninfected cells were seeded (5 × 10⁴ cells per well) in 8-well chamber slides (Becton–Dickinson, Franklin Lakes, NJ, USA) and then infected as described above. After infection, cells were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min, permeabilized with 0.1% Triton X100 in PBS, and saturated with 3% bovine serum albumin (BSA), 0.1% Tween 20 in PBS. For staining, cells were incubated overnight with a human serum containing IgG to SARS-CoV-2 (1:1000 dilution), with an anti-SARS-CoV-2 spike (S) glycoprotein monoclonal antibody (1:1000 dilution; abcam, clone 1A9, Cambridge, UK), anti-SARS-CoV-2 nucleocapsid (NP) monoclonal antibody (1:1000 dilution; abcam, clone 6H3), anti-SARS-CoV-2 envelope (E) polyclonal antibody (1:1000 dilution; abcam) followed by Alexa Fluor 488-conjugated anti-human, mouse or rabbit IgG (Thermo Fisher Scientific). Nuclei were counterstained with 4′,6-diamidino,2-phenylindole (DAPI, Merck, Darmstadt, Germany). Cells were photographed under a Zeiss Axiovert 200 M epifluorescence microscope equipped with a Plan-Apochromat 40x or 63x/1.4 NA oil objective. Z-stack images acquired using the ApoTome imaging system were elaborated through the AxioVision Extended Focus module (Zeiss Axiovert 200 M system).

GM7373-VEGFR2 (VEGFR2-ECs) cells were seeded on VEGF-A or gremlin or uncoated coverslip for 4 hours and then incubated with soluble gremlin (150 ng/mL) for 90 minutes at 4 °C in PBS added with Ca2+ and Mg2+ and 0.1% gelatin. Then, cells were washed three times with PBS or with PBS plus 1.5 mol/L NaCl to remove HSPGs-bound ligands. Immunofluorescence analysis was performed using a goat polyclonal anti-gremlin antibody (R&D Systems) or goat polyclonal anti VEGF-A antibody followed by AlexaFluor 488 anti-goat IgG (Molecular Probes, Life Technologies). Cells were analysed using a Zeiss Axiovert 200 M system.

The transgenic cells were seeded in a 6-well plate and cultured until cell colonies formed. Then, the cells were fixed with 4% paraformaldehyde (Sigma, USA) for 15 min at room temperature and permeabilized with 0.25% TRITON™ X-100 (Promega, USA) in DPBS for 15 min. Subsequently, the cells were blocked with 1x blocking solution (DaeMyung Science, Korea) for 1 h at room temperature and incubated with anti-Nanog (1 : 500 dilution; Abcam, UK) or anti-Oct4 (1 : 50 dilution; Abcam, UK) at 4°C in 1x blocking solution overnight. After washing three times with 1x phosphate-buffered saline with Tween 20 (LPS solution, Korea), the cells were incubated with AlexaFluor® 555-conjugated secondary antibodies (Invitrogen, USA) for 2 h at room temperature. Finally, the nuclei were stained with 5 μg/mL Hoechst 33342 solution (Sigma, USA) for 5 min at room temperature. Cell images were acquired using an Axiovert 200M system (Carl Zeiss, Germany).