Dmi3000 m inverted manual microscope

To assess the migration ability of ADSCs, a Transwell migration assay using a Transwell system from Corning (Corning, Inc., Corning, NY, USA) was performed. Specifically, A375 cells were seeded onto the plastic surface of the 24-well plates at a density of 1×10⁵ cells/well and grown overnight, while HFF cells were used as a control. On the following day, parental ADSCs and TRAIL-ADSCs were seeded on the top chambers of Transwells, respectively and cultured in serum-free medium for 48 h at 37°C and in 5% CO₂ in an incubator. At the end of the experiments, the up-chamber was washed with PBS and scraped gently using a cotton swab to remove non-migrated cells. The ADSCs migrated to the bottom side of the filter and were fixed with 70% ethanol, stained with 0.1% crystal violet (#C0121; Beyotime Institute of Biotechnology) and counted under a DMI3000 M inverted manual microscope (Leica Microsystems GmbH). The average number of migrated cells was assessed by counting five randomly selected microscopic fields. The experiment was performed in triplicate.

Transwell inserts of 8 µm-pore plain (to assess migration) or Matrigel-coated (to assess invasion; Costar; Corning, Inc.) were placed in the wells of 24-well culture plates and 500 µl of DMEM or RPMI-1640 containing 10% FBS was added to the lower chamber. The T29H, OVSAHO, or SKOV3 cells were washed once with Hanks' Balanced Salt Solution (Invitrogen; Thermo Fisher Scientific, Inc.) 12 h after transfection, resuspended in 100 µl serum-free medium (8×10⁴ cells) and added to the upper chamber. After 12 h of incubation at 37°C with 5% CO₂, the cells on the top side of the filter were manually removed with a cotton swab. The cells adherent to the bottom surface of the insert were fixed in cold absolute methanol for 10 min and then stained with 0.01% crystal violet in 20% ethanol at room temperature. After 10 min, the filters were washed thoroughly in water and images were captured under a DMI3000 M inverted manual microscope (Leica Microsystems GmbH). The number of migratory cells was recorded using an optical microscope at ×100 magnification. The average number of migrated cells was assessed by counting five randomly selected microscopic fields. The experiment was performed in triplicate.