Eclipse ts2

To generate CSC-rich population, A549 cells were grown in a 24-well ultralow attachment plates at a density of 1.5 × 10³ cells/well in DMEM medium containing 1% FBS for 7 days to form primary spheroids. At day 7, primary spheroids were harvested and resuspended as single cells using 0.25% trypsin–EDTA (HyClone) and seed onto 24-well ultralow attachment plates for 14 days to form secondary spheroids. After 14 days of secondary spheroid development, each secondary spheroid was collected, dissociated into a single spheroid of the same size, and treated with non-cytotoxic concentrations (0 − 2.5 µM) of RES or MOS for 3 days. Phase-contrast images of secondary spheroids were captured (day 0, 1, and 3) after treatment using a phase-contrast microscope (Nikon ECLIPSE Ts2). At day 3, a single spheroid was co-stained with Hoechst 33342 and PI at 37 °C for 15 min and photographed under a fluorescence microscope (Nikon ECLIPSE Ts2).

Medulloblastoma cells were seeded in 25 cm² flasks (100,000 cells), left to attach, and treated with RKI-1447 (IC₅₀ from the viability assay 72 h and half of the IC₅₀ value), and images were acquired with a phase-contrast microscope Nikon Eclipse Ts2, 20× objective (Nikon Instruments Inc., Melville, NY, USA).

The animal ethics committee of Guangzhou Red Cross Hospital approved the research. Following previously published methods (28 ), a 3 cm × 3 cm wound was made on the back skin of the rats. The wound was disinfected every day, and the rats were provided with enough food and water to ensure their normal activities and survival. After 1 week, 0.8% pentobarbital was injected into the abdominal cavity of these animals for anesthesia. After anesthetization, approximately 5 mL of abdominal aortic blood was collected using a fine-needle approach, and the blood sample was diluted to 1:1 by PBS. Mononuclear cells (MNCs) were separated and collected with Ficoll separation solution (GBCBIO Technologies, Guangzhou, China) and centrifuged at 2000 rpm for 35 min. The middle layer was pipetted with a thin tube and washed twice with phosphate-buffered saline (PBS). MNCs (2×10⁶/mL) were seeded onto the T-25 flask with 10 mL of complete Dulbecco’s Modified Eagle Medium (Gibco, MA). The complete medium contained 1% penicillin/streptomycin (Gibco), 20 ng/mL bFGF (R&D Systems, MN), and 20% fetal bovine serum (Gibco). With 21 days of culture, the cell convergence was 80%, and the third-generation cells digested by 0.25% trypsin were used for subsequent experiments. Representative bright-field images were captured by an inverted phase-contrast microscope (Nikon ECLIPSE Ts2, Nikon).

The invasive potential was determined using a 48-well micro chemotaxis Boyden chamber (Neuro Probe, Cabin John, MD, USA). HepG2 and HCCLM3 cells were propagated in a Boyden chamber containing polycarbonate membrane (8-mm pore size) which has been coated with matrigel. Thereafter, the Boyden chamber assay was carried out as reported in our prior reports [59 (link),60 (link)]. The invading cells on the membranes were observed by a Nikon ECLIPSE Ts2 (Nikon corporation, Tokyo, Japan).

After 72 h of treatment, the medium was removed and the non-adherent cells were gently removed, washing with PBS 1x. Dish-anchored cells were counted using the inverted microscope Nikon ECLIPSE Ts2 [37 (link)]. TPA 50 nM was used as the positive control.

The assay was performed as described by Đorđevski et al. [49 (link)] with some modifications. HaCaT cells were grown until reaching 85% confluence. The cell monolayer was scratched with a 200 μL sterile tip. Floating cells were washed, and cells were incubated in reduced DMEM supplemented with 1% FBS, 2 mM L-glutamine, and 1% antibiotic-antimycotic, containing 400 µg/mL of preparations. Cell migration was monitored using Nikon Eclipse TS2 (Amsterdam, the Netherlands) 48 h after wound preparation and treatment. The untreated control was used to measure wound closure under these conditions and without the addition of preparations. Results were presented as the percentage of wound closure during exposure to the tested extracts.