Ec3 camera

A549 cells were cultivated in a 6-well plate containing DMEM culture medium that is supplemented with 1% penicillin–streptomycin and 10% FBS under 5% CO₂ with balanced humidifier air and 37 °C. The next day, when the cells attached and spread on the culture surface, the conditioned culture medium was replaced with fresh media containing 0.02 mg/mL biotin-X DHPE and incubated for an additional 24 h. To confirm biotinylation, the media were discarded, and cells were incubated for 1 h in streptavidin PE-Cy5.5 to bind the streptavidin with the biotinylated cell surface. Then, the cells were gently washed with PBS to discard any unbounded streptavidin PE-Cy5.5. Phase-contrast along with fluorescence images of cells were taken with a Leica DMi8 microscope equipped with a Leica EC3 camera (Leica Microsystems, Wetzlar, Germany). For tethering MYR-coreSA fusion protein on the surface of A549 cells, the biotinylated media were discarded, and the fusion protein (2 µg) was incubated for 1 h, followed by a gentle wash with 1× PBS to discard unbounded fusion protein.

Sections were photographed with a Leica EC3 Camera (Leica microsystems, Switzerland) connected to a Zeiss Axioscope 40 microscope (Carl Zeiss, Germany) with the Leica Application suite Version 4.6.0 (Leica microsystems, Switzerland). All image analysis was prepared with Image 1.50i software (National Institutes of Health, USA).

HMVEC-dLy grown up in T75 flasks to near confluence (80%) were trypsinized and resuspended in endothelial basal media (EBM) (without any growth supplements) to a final concentration of 2×10⁵ cells/ml. Growth Factor Reduced Matrigel (BD Biosciences, Mississauga, ON, Canada) was thawed overnight at 4°C, diluted with cold EBM (1:1 dilution) and then a volume of 300 μl Matrigel solution/well was placed in six-well plates to solidify for 2 hours. After that, HMVEC-dLy cells (2 ml of cell suspension) were seeded on the solidified Matrigel under various concentrations of CCL21/6Ckine (0, 100, 200, and 350 ng/ml) for 24 hours. Tube formation was examined on an inverted microscope (100× magnification) at different time intervals. Images were randomly taken with Leica EC3 camera (Leica Microsystems) in different areas of the wells by selecting fields of view that were distinct and distant enough to not overlap with each other. The total length of the interconnected cells forming tubular structures was measured with ImageJ.

HEK293T cells were seeded at 1.7 × 10⁵ cells/cm² within 10 mL of DMEM with 10% FBS and 1% penicillin-streptomycin in a T-75 flask. The next day, 5 µL of pcDNA3.1-hACE2 plasmid (Addgene) was mixed with 0.1 mL of PEI for 30 min and then added to the cells for transfection. The culture medium was replaced with a fresh one. After 48 h, 500 µg/mL G418 disulfate (Sigma-Aldrich) was used for selection for up to four weeks. The stable HEK293T cell line expressing hACE2 (HEK293T-ACE2) on the surface of the outer cell membrane was confirmed by incubating HEK293T-ACE2 cells with anti-ACE2 mAb for 1 h at room temperature. Following three gentle PBS washes, cells were treated for 25 min at room temperature with a fluorescent secondary antibody: anti-mouse IgGκ binding protein conjugated with red fluorescent dye CruzFluor^TM 594 (Santa Cruz Biotechnology). A Leica DMi8 microscope equipped with a Leica EC3 camera (Leica Microsystems, Wetzlar, Germany) was used to image the cells. Western blot analysis was performed in HEK293T-ACE2 cells by using RIPA lysis buffer (Thermo Fisher Scientific). Briefly, after performing SDS-PAGE for the cell lysate, the gel was transferred onto the nitrocellulose membrane. ACE2 mAb and HRP-conjugated mouse IgG secondary antibodies were used for Western blot detection.

Invasion assays were performed as described elsewhere [10 (link)]. Cells were grown in 0.2% FAF-BSA-containing serum-free medium (SFM) overnight before the start of an experiment. In some experiments, cells were pre-incubated with JTE-013 (10 μM, 1 h), VPC 23019 (1 μM, 1 h), C3-Transferase (100 ng/ml, 4 h), Y-27632 (10 μM, 1 h), SB-3CT (10 μM, 1 h) or ALLN (50 μM, 1 h). The inhibitors were present in both the upper and lower chambers throughout the experiment. The cells were stimulated with S1P and allowed to migrate towards 5% lipid-stripped FBS (LS-FBS) for the time points indicated and then non-migrated cells were removed with a cotton swab. The migrated cells were fixed in 2% paraformaldehyde for 10 min and then stained with 0.1% crystal violet in 20% methanol for 5 min. The membranes were washed with PBS and water and allowed to dry overnight. The cells were counted at 40X magnification in eight to ten fields in a straight line bisecting the membrane. Images of invasion inserts were captured using Leica EC3 camera (Leica Microsystems, Switzerland Ltd., CH, Switzerland) and with 20X magnification objective on Leica microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany).