Eclipse te2000 u light microscope

A single cell suspension (3 × 10³ cell/well) and 1X Spheroid Formation ECM (Cultrex^®, Trevigen Inc., Gaithersburg, MD, USA) (5 µL/well) was prepared, and 50 µL of this suspension was placed in each well of the 96-well ultra-low attachment Costar^® plates (Corning Inc., Corning, NY, USA). The plate was centrifuged at 200× g for 3 min and incubated in a 37 °C, 5% CO₂ incubator for 72 h. Then, the plate was left on ice for 15 min, and 50 µL of Invasion Matrix (Cultrex^®) was added to each well (day 0). The plate was then centrifuged at 300× g, 4 °C, 5 min, followed by a 1 h incubation. The spheres were then treated with different concentrations of Rg3. At day 0 and day 7, images were taken of the spheres using a Nikon Eclipse TE2000-U light microscope, and the area of each sphere was measured using NIS-elements software (Nikon, Tokyo, Japan). The invasion (%) of each spheroid was normalized to the mean invasion area of the vehicle group.

Cellular migration was measured to assess antimigratory effect of bac I and II either alone or in combination on HT-29 by circular scratch wound migration assay as described previously [13 (link),57 (link)]. Briefly, HT-29 cells were seeded in a complete medium at 1 × 10⁵ cells per well on 96-well flat-bottomed plates and incubated under the standard culture condition until 80% confluent. The medium was replaced with a serum-reduced medium and cells were incubated overnight. A circular wound was made on the cellular monolayer using a 10 µL pipette tip. The serum-reduced medium was changed to a complete medium, supplemented either with vehicle or bac I and/or II, and, to inhibit cell proliferation, 1 μg/mL mitomycin C (Sigma-Aldrich, St. Louis, MO, USA). Images were captured at time 0 and after 48 h on an Eclipse TE2000-U light microscope (Nikon, Tokyo, Japan) at 40× magnification. NIS-Elements BR software (Nikon) was used to measure the area of the wound relative to the area at time 0.

Cells were plated on 96-well plate in the respective complete medium at 1.2 × 10⁴, 4 × 10⁴ and 3.5 × 10⁴ cells per well for 2H-11, 3B-11 and HUVEC lines, respectively. After 6 h of incubation in 5% CO₂ at 37 °C, the medium was replaced with DMEM-based media supplemented with 2% FBS for 2H-11 and 3B-11 lines and HUVEC growth medium for HUVEC line, both containing 1 µg/mL mitomycin C (Sigma-Aldrich). These media are referred to as “mitomedia”. A scratch wound was made on the cell monolayer and cells were incubated for 1 h in the fresh mitomedia. The medium was then changed to mitomedia containing various concentrations of compounds with DMSO as a vehicle. Area surface of the wound was serially monitored and images were obtained for the next 16 h on Eclipse TE2000-U light microscope (Nikon, Tokyo, Japan) at 40× magnification. The wound area was quantified using NIS-Elements BR software (Nikon) and the result was expressed as relative wound closure (%) compared to time point 0.