Eclipse ts2r fl

T24 or HUVEC cells were seeded in 24-well plates and washed with PBS, before being fixed with 4% paraformaldehyde. Then, cells were dyed by 4’,6-diamidino-2-phenylindole (DAPI) for 5 min in the dark. Cells were observed with an excitation wavelength of 358 nm and an emission wavelength of 461 nm using a fluorescence spectrophotometer (ECLIPSE Ts2R-FL, Nikon, Japan).

The cells were cultured in 96-well plates at a density indicated above and treated with the compounds under study. The cells were incubated for 24 h, after which they were washed with HBSS and fixed with a 10% formalin solution for 20 min at room temperature. Phalloidin–CF543 (5 U/mL) and DAPI (0.25 µg/mL) were added, and the cells were stained for 30 min at room temperature.
Images were acquired in an inverted Eclipse Ts2R-FL (Nikon) equipped with a Retiga R1 camera and treated with Fiji.

Human hepatoma cells (HepG2 cells) were cultured in DMEM high glucose medium, supplemented with 10% inactivated FBS, 2 mmol/L L-glutamine, and 1% penicillin-streptomycin. Cells were maintained at 37 °C in a humidified 5% CO₂ atmosphere; medium was replaced every two days until cells were confluent. The cytotoxic activity was assessed using an MTT assay [42 (link)]. Cells were seeded in 96-well plates at a density of 1 × 10⁶ cells/well and cultured for 12 h. 0.1 mg/mL β-glucosidase, and 2, 4, 6, 8, and 10 mg/mL of D-amygdalin or L/D-amygdalin (isomer ratio 1) were dissolved in DMEM medium and aliquoted onto cells. Cell viability was calculated using the following formula:
The median effective doses (IC₅₀) of D- and L/D-amygdalin were calculated using GraphPad Prism 8. The morphology of each treatment group was observed using an inverted fluorescence microscopy (Nikon ECLIPSE Ts2R-FL, Tokyo, Japan).

ECFCs migratory function was assessed in transwell migration assay as described by Tsai et al.^{2 (link)} with minor modifications. Briefly, EGM-2 medium supplemented with 10% FBS was added into the lower chamber, while 5 × 10⁴ cells in endothelial basal media (EBM-2) were seeded onto upper chamber of transwell permeable supports (8 µm pore size, polycarbonate membrane, SPL Life Sciences). Thereafter, cells were incubated for 4 h at 37 °C, 5% CO2. Suspension cells remaining in the upper chamber were removed and the migrated cells present in the lower side of the transwell insert were fixed with 4% paraformaldehyde. The migrated cells were next stained with DAPI and images from random selected fields and counted number of migrated cells using inverted fluorescence microscope (Nikon ECLIPSE Ts2RFL) and number of migrated cells were quantified and evaluated by Image J software (version 1.51 J8; National Institute of Health, USA).

The cytotoxicity of SDT was evaluated by CCK-8 assay. After the completion of incubation and irradiation described previously, the cells were further incubated for another 2 h. The cell viability was assessed using CCK-8, and the OD 450 nm value was measured by a multimode reader (Synergy HTX, BioTEK Co., USA). Additionally, the cell viability was evaluated by live/dead cell staining. Similarly, the treated MC38 cells were stained with Calcein-AM and PI for 30 min, and observed by fluorescence microscope (ECLIPSE TS2R FL, Nikon, Japan). CRT expression on the treated cell surface was also observed to assess immunogenic cell death caused by SDT. Briefly, after fixation with 4% paraformaldehyde, the treated cells were blocked with 5% BSA, incubated with the anti-calreticulin antibody overnight and then with secondary antibody for 1 h. After further counterstaining with DAPI, the MC38 cells were visualized by fluorescence microscope. Semi-quantitative analysis of the fluorescence intensity was performed using Image J software.

To analyze the PPARγ intracellular localization, mouse B16F10 cells were treated with or without CLQ (1.5 μM, an effective dose in inhibiting proliferation, migration, and glycolysis in B16F10 cells) for 24 h. After that, cells were fixed with ice-cold 4% paraformaldehyde for 30 min, followed by blocking in 5% goat serum for 1 h, and incubation with rabbit polyclonal anti-PPARγ antibody overnight at 4 °C. After repeated washing, the cells were probed with secondary antibodies conjugated to Alexa Fluor 488 anti-Rabbit IgG (Cat. No. 4408 S, 1:500 dilution, Cell Signaling Technology) for 1 h at room temperature. Nuclei were identified with DAPI. The sections were photographed with a Nikon fluorescence microscope (ECLIPSE, Ts2R-FL, Tokyo, Japan).

Detection of the late apoptosis of CPAE cells induced with LPS after treatment with C. nutans extract was investigated using TUNEL assay, using the DNA Fragmentation Imaging kit (Merck, Darmstadt, Germany). CPAE cells induced with LPS were treated with C. nutans extract (25, 50, and 100 µg/mL) for 24 h. After incubation, the cells were washed with phosphate buffer saline (PBS, pH 7.4) three times. The cells were then fixed with 100 µL of 4% paraformaldehyde and incubated at room temperature for 10 min. One hundred microlitres of 0.1% Triton-X100 was added after removing the fixing solution and the cells were then incubated at room temperature for 20 min. After washing twice with PBS, the solution of terminal deoxynucleotidyl transferase (TdT) enzyme was added and the cells were incubated at 37 °C for 1 h. Then, nuclei dye mixture solution (Hoechst) was added and the cells were further incubated in the dark at room temperature for 15 min. The solution was removed, and the cells were mounted with ProLongTM gold antifade mountant (Life technologies, Camarillo, CA, USA) before detection using an inverted fluorescence microscope (ECLIPSE Ts2R-FL, Nikon, Tokyo, Japan).