Transwell insert system

Cell migration and invasion were analyzed in vitro using the Transwell insert system (Corning, United States) with or without Matrigel coating (BD, United States), respectively. Medium (600 μL) containing 10% FBS was added outside of the Transwell culture insert. For CTRL group, 100 μL of serum-free medium containing 2 × 10⁴ cells was added to each well of the insert. For RT group, cells were treated with irradiation followed by serum-free medium, then added to the insert. For MSC group, cells treated with serum-free AT-CM were added. For RTM group, cells were treated with irradiation followed by serum-free AT-CM. After incubation for 24 h, the Transwells were washed and cleaned using cotton swabs and then fixed with 100% methanol for 15 min, washed twice with PBS, stained with 0.1% of crystal violet for 10 min, and observed under a microscope (Leica, Germany). The experiment was performed in triplicate.

After treated, cells were trypsinized and counted. Cell migration and invasion were analyzed in vitro using the transwell insert system (Corning) without coating or with coating by 20 μL of Matrigel (BD Biosciences, USA), respectively. The culture insert was attached on the bottom of a 24‐well plate, and 100 μL of serum‐free media containing 1 × 10⁵ cells was seeded into each well of the insert. Six hundred μL of media containing 10% FBS was added outside the transwell culture insert. Cells were incubated at 37°C for 18 and 24 hours in a humidified atmosphere with 5% CO2 for migration and invasion, respectively. Transwells were cleaned using cotton swap. The cells were fixed with 1% formaldehyde for 15 minutes, washed twice with Phosphate buffered saline (PBS), stained with 0.1% of crystal violet for 15 minutes, washed with distilled water, and then observed using a microscope (Leica, Wetzlar, Germany).

Cell migration and invasion were examined using a 24-well Transwell insert system (Corning, NY). For cell migration assay, cells (1.5 × 10⁶ cells/mL) were cultured in the upper chamber-containing FBS-free DMEM at 37 °C with 5% CO₂ for 24 h. The lower chamber was supplemented with DMEM and 10% FBS. The migrating cells on the below side of chamber were collected. After 95% alcohol of fixation, the migrated cells were stained with 1% crystal violet (Solarbio) for 5 min. Finally, five random visual fields were selected to observe and analyse the differences among groups under an inverted microscope (Olympus, Tokyo, Japan).
For cell invasion assay, matrigel (Becton Dickinson Biosciences, San Diego, CA,) was covered on the basolateral Transwell chambers. Except for this step, cell invasion assay had the same steps with cell migration assay.

Cell migration and invasion were analyzed in vitro using the transwell insert system (Corning, USA) without coating or with coating by 20 μL of Matrigel (BD, USA), respectively. The culture insert was attached on bottom of a 24-well plate, and 100 μL of serum-free media containing 1.5 × 10⁵ cells were seeded into each well of the insert. Six-hundred μL of media containing 10% FBS was added outside the transwell culture insert. Cells were incubated at 37°C for 18 hrs and 24 hrs in a humidified atmosphere with 5% CO₂ for migration and invasion, respectively. Transwells were washed twice with PBS and cleaned using cotton swap. The cells were fixed with 1% formaldehyde for 15 mins, washed twice with PBS, stained with 0.1% of crystal violet for 15 mins and then observed using a microscope (Leica, Germany).