Inverted microscope

A scratch assay was used to evaluate cellular migration. The VSMCs were cultivated in 12-well plates before being scraped with a pipette tip. After being washed with PBS (Cat. No. BCBS2233V, Sigma-Aldrich, Germany), the cells were cultured in the medium containing dexamethasone (10^–7, 10^–6, and 10^–5 M) for 24, and 48 h. Images of the injured area were obtained using an inverted microscope (OPTIKA, Italy). Image J software was used to perform image analysis.

Any alterations in the morphology or growth of the treated cells were observed under an inverted microscope (Optika) at 24 and 48 h.

Approximately 5 × 10⁴ U-87 MG cells were cultured in 24-well plates and placed inside the incubator until a confluent monolayer formed. After 24 to 48 h of incubation and the cells adhering to the bottom of the plate, a scratch was created. A sterile plastic micropipette tip was used to simulate an in vitro wound by creating a straight-edged, cell-free zone across the cell monolayer in each well. Following the scratch, the monolayer was washed with PBS, and DMEM culture medium enriched with 10% FBS was added, then returned to the incubator. The migration progress was documented by capturing sequential digital photographs of the gap using an inverted microscope (OPTIKA, Italy) at 0, 48, and 72 h after scratching. A reasonable approach is to capture three images per well per time point. The amount of cell migration was calculated by measuring the distance between the two edges of the scratch using the public domain software ImageJ, and data analysis was performed using IBM SPSS Statistics 27.0.

EJ cells were plated in 6-well plates and grown to 90% confluence in 2 mL of growth medium and a line-shaped incision to the confluent monolayer of growth-arrested cells was generated using a 2 mm pipette tip. The cells were treated with various concentrations of SPHF for 24 h and allowed to migrate into the scraped area. Images were captured using an inverted microscope (40 x magnification; Optika, Ponteranica, Italy).

MDA-MB-231 and 3T3L fibroblasts were treated with the optimal dose of 5% of T1 and were observed under the inverted microscope (OPTIKA Microscopes, Ponteranica, Italy) after 24 and 48 h of exposure to the treatment.

To determine the effect of T1 extract treatment of the invasive capacity of the BC cell, cell invasion assay was performed using the Boyden Matrigel Chamber assay. Cell invasion assay was carried out in 24-well Biocoat Matrigel invasion chambers (pore size of 8 μm, Corning, NY, United States) according to the manufacturer’s protocol. Briefly, untreated (control) and treated (5% of T1 extract as well as methanol) MDA-MB-231 cells were placed onto the upper chambers of Matrigel plates, and the bottom chamber was filled with DMEM medium containing 10% FBS as chemoattractant, and then incubated at 37°C. After 48 h incubation, the upper chambers were washed with sterile PBS and non-invasive cells were removed gently using a sterile cotton swab. Cells that invaded to the lower surface of the membrane were fixed with methanol and formaldehyde for 10 min and stained with 0.5% crystal violet. After washing out the stain with PBS, invaded cells were photographed under the inverted microscope (OPTIKA Microscopes, Ponteranica, Italy) in five predetermined fields. Percentage inhibition of invasive cells was calculated with respect to untreated cells and quantified using Image J software. Each experiment was carried out in triplicates.