Bz x710 fluorescent microscope

Tube formation assays were conducted using EA.hy926 cells and Matrigel matrix (Corning Inc.) in the presence of the supernatants of pancreatic cancer cells. Cell supernatants were collected from the cancer cell culture medium (MIA PaCa-2 and SW-1990 cells were cultured in DMEM and BxPC-3 cells were cultured in RPMI medium) via centrifugation at 400 × g for 5 min at 14°C. MIA PaCa-2, SW-1990 and BxPC-3 cells were seeded at 4×10⁵ cells/well in six-well plates containing medium (MIA PaCa-2 and SW-1990 cells were cultured in DMEM and BxPC-3 cells were cultured in RPMI medium) and cultured for 1 day at 37°C. The culture media were then changed, and the cells were incubated for an additional 48 h at 37°C in medium with or without 10Z-Hymenialdisine (5 µM), after which the supernatants were collected and centrifuged at 400 × g for 5 min at 14°C to remove particles. EA.hy926 cells (1.2×10⁴ cells/well) were seeded into each well of a 96-well plate coated with Matrigel. Matrigel was pre-coated for 30 min at room temperature. Then, the cells were cultured for 1 day at 37°C with 50 µl 10% FBS and 50 µl of the aforementioned supernatant. Tube formation was observed by a BZ-X710 fluorescent microscope at ×40 magnifications (Keyence Corporation) and evaluated by determining the number of the endotubes generated by EA.hy926 cells. A total of four random fields of view were analyzed per sample.

The day before infection, 1 × 10⁴ Vero E6 cells per well were seeded in an eight well chamber slide (Nunc Labtek chamber slides, Thermoscientific). On the day of infection, SARS‐CoV‐2, isolate USA‐WA/2020 (BEI resources, cat no. NR‐52281) was added to cells at an MOI of 0.01. The infection was carried out for 48 h. Cell layers were washed and incubated with 8 μg/ml A568‐conjugated monoclonal antibody for 30 min at room temperature (RT), washed and fixed in 10% neutral‐buffered formaldehyde for 30 min at room temperature (Duke GHRB SOP 38, attachment 21). After two washes with Wash Buffer (1%FBS‐PBS; WB), cells were permeabilized with 0.5% Triton for 5 min at RT. Blocking buffer was added and the cells were incubated at 4°C for 4 h. After washing, 10 μg/ml anti‐SARS‐COV‐2 nucleocapsid antibody (40143‐MM08, Sino Biological), conjugated with A488 using an Alexa fluor antibody labelling kit (Invitrogen) was added and incubated at 4°C overnight with rocking. Cells were then washed and Fluorshield DAPI reagent (Sigma) added before the cover slip was mounted on the slide. Cells were visualized using a Keyence BZ‐X710 fluorescent microscope and images analyzed using BZ‐X Analyzer software (v1.3.11).

Infected cells were prepared as described above. On the day of assay, Cell layers were washed and incubated with plasma from infected individuals diluted at 1:100 for 30 min at room temperature (RT), washed and fixed in 10% neutral‐buffered formaldehyde for 30 min at room temperature (Duke GHRB SOP 38, attachment 21). After two washes with Wash Buffer (1%FBS‐PBS; WB), cells were permeabilized with 0.5% Triton for 5 min at RT. Blocking buffer was added and the cells were incubated at 4°C for 4 h. After washing, 10 μg/ml anti‐SARS‐COV‐2 nucleocapsid antibody (40143‐MM08, Sino Biological), conjugated with A568 using an Alexa fluor antibody labelling kit (Invitrogen), was added and incubated at 4°C overnight with rocking. After washing, A488‐conjugated anti‐Human IgG Fc antibody (Clone: HP6017, Biolegend) was added and incubated for 30 min at 4°C on a rocking platform. Cells were then washed and Fluorshield DAPI reagent (Sigma) added before the cover slip was mounted on the slide. Cells were visualized using a Keyence BZ‐X710 fluorescent microscope and images analyzed using BZ‐X Analyzer software (v1.3.11).

MIA PaCa-2, SW-1990 and BxPC-3 cells were seeded at 1×10⁴ cells/chamber in a four-chamber glass slide and cultured for 1 day at 37°C. The cells were treated with 10Z-Hymenialdisine (5 µM) for 2 h at room temperature, followed by stimulation with TNF-α (1 ng/ml) for 20 min. Cells that had not been treated were used as controls. The cells were then washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Next, the cells were washed with PBS, permeabilized with 0.1% Triton-X for 3 min, and incubated with 3% bovine serum albumin (BSA; FUJIFILM Wako Pure Chemical Corporation) for 1 h at room temperature. Subsequently, slides were treated with rabbit antibodies against NF-κB p65 (Cell Signaling Technology, Inc.; cat. no. 8242; 1:400) overnight at 4°C and then treated with goat anti-rabbit IgG secondary antibodies H&L (Alexa Fluor^® 488) (cat. no. ab150077; Abcam) for 1 h at room temperature. Primary and secondary antibodies were used at 1:400 and 1:500 dilution with 3% BSA, respectively. Cell nuclei were subsequently stained with DAPI (50 ng/ml) at room temperature for 10 min. Images of the stained slides were captured using a BZ-X710 fluorescent microscope at ×400 magnification (Keyence Corporation).