Eclipse ti s inverted microscope system

HUVEC-CS cells were seeded in 6-well plates and grown as a monolayer in DMEM supplemented with 20 µM TP1 (or the PBS control) under normoxic or hypoxic conditions for 24 h. The ginsenosides Rb1 and Rg1 were used as reference controls at 20 µM. THP-1 cells were cultured in RPMI medium supplemented with 10% FBS. Immediately prior to their use in adhesion assays, the THP-1 cells were harvested, washed three times with PBS, and labeled with 10 µM carboxyfluorescein succinimidyl ester (CFSE) for 10 min at 37 °C. The cell culture medium was aspirated from the HUVEC-CS monolayers, which were then washed with a serum-free RPMI medium. CFSE-labeled THP1 cells were then seeded onto the HUVEC-CS monolayer at a density of 2 × 10⁵ cells/well in serum-free RPMI medium and incubated for 90 min at 37 °C. After incubation, the culture medium was drained, and the HUVEC-CS monolayer was washed three times with PBS to remove nonadherent leukocytes. Images of adherent leukocytes were then captured using the green fluorescence channel of a Nikon ECLIPSE Ti-S inverted microscope system (Nikon, Yokosuka, Kanagawa, Japan). Cells were quantified via cell counting, and statistical analyses were performed using data collected in triplicate experiments.

HOS-eGFP-LC3 and A549-eGFP-LC3 cells were infected with viruses at MOI 10 for 48 h (n = 3, biological replicates). LC3 accumulation in autophagosomes were visualized in an eclipse Ti-S inverted microscope system (Nikon, Tokyo, Japan) equipped with a Zyla sCMOS camera (Andor Technology, Belfast, Northern Ireland).

Thioflavin-T staining was used to detect UPR activity. Cells were grown on coverslips under various experimental conditions for 24 h, washed with PBS, and fixed with 4% paraformaldehyde for 20 min at room temperature. The fixed cells were then permeabilized for 5 min with 0.2% Triton X-100 at room temperature, stained with 1 μg/mL DAPI for 5 min, and counterstained with 5 μM thioflavin-T for 10 min. Coverslips were then mounted on glass slides with Clarion mounting medium, and images were captured using the Nikon ECLIPSE Ti-S inverted microscope system. Quantification of fluorescence intensity was performed by analyzing labeled cells in ImageJ. The experiment was performed in triplicate.

Intracellular ROS activity was measured using a DCFDA-based fluorogenic assay. Briefly, ECs were grown as a monolayer for 24 h under various experimental conditions, washed with serum-free medium, and stained with 1 μM DCFDA for 60 min at 37 °C. The dye-containing medium was then replaced with a fresh medium, and images of DCFDA-labeled cells were captured using the green fluorescence channel of the Nikon ECLIPSE Ti-S inverted microscope system. Quantification of fluorescence intensity was performed by analyzing labeled cells in ImageJ. Statistical analyses were performed using data from triplicate experiments.

Glucose uptake was measured using 2-NBDG, a fluorescently labeled deoxyglucose analog. Pre-cultured cells were washed twice with serum- and glucose-free DMEM before incubation in serum- and glucose-free DMEM containing 50 µM 2-NBDG for 30 min at 37 °C, followed by washing with ice-cold PBS. The 2-NBDG that was taken up by the cells was extracted by lysing cells in lysis buffer [1% Nonidet P-40, 1% sodium deoxycholate, 40 mM KCl, and 20 mM Tris (pH 7.4)]. Fluorometric quantification was performed using a microplate reader (Tecan Magellan™, Switzerland) with excitation and emission wavelengths of 467 nm and 542 nm, respectively. Protein content was measured with a BCA protein assay to normalize fluorometric results. For microscopy, after incubation with 2-NBDG, the cells were washed with ice-cold PBS and placed in Hank's balanced salt solution (HBSS). Images were captured using the green fluorescence channel of a Nikon ECLIPSE Ti-S inverted microscope system (Nikon, Kanagawa, Japan). Assays were performed in triplicate.