Diff quick kit

Invasion assay was performed using Matrigel-transwell chambers (8 μm pore). Invasion assay was performed using Matrigel (BD Biosciences, San Jose, CA, USA)-coated transwell chambers (Corning, Corning, NY, USA). Cells (2.5 × 10⁴ cells/well) were placed in the upper transwell chamber, and medium containing 0.1% BSA was added to the lower chamber. The next steps were done according to the manufacturer’s instructions. After incubation for 16 h, the cells that migrated to the lower surface of the filter were fixed and stained using Diff-Quick kit (Fisher Scientific, PA, USA). The stained cells were counted under a light microscope (Mitoti AE31 series, Trinocular Inverted MIC).

For invasion assay, breast cancer cells were loaded in Transwells with 8 μm pore size filter inserts (Corning Glass, Seoul, Korea) that were precoated with 10 mg/mL growth factor-reduced matrigel (BD Biosciences, Seoul, Korea) on the upper side of the chamber with the lower well filled with 0.8 ml of growth medium. After incubation for 48 h at 37 °C, and For migration assay, we used Transwells with inserts that contained the same type of membrane but without the matrigel coating. After incubation for 24 h at 37 °C. Non-invaded cells on the upper surface of the filter were removed with a cotton swab and migrated cells on the lower surface of the filter were fixed and stained with the Diff-Quick kit (Fisher, Pittsburgh, PA, USA) and photographed (magnification ×20). Invasiveness and motility were determined by counting cells in four microscopic fields per well, and the extent of invasion was expressed as an average number of cells per microscopic field. Cells were imaged by phase-contrast microscopy (Leica Microsystems, Bannockburn, IL). All experiments were repeated three times.

For scratch assays, OACs were cultured in 24-well tissue culture dishes to 80% confluency. A scratch of ≈0.4–0.5 mm was made using a sterile 10 µL pipette tip. Each well was washed three times with particle-free PBS to remove residual cells prior to addition of serum-free medium for control, and SFM + MSC-EV (1:4 ratio OAC:MSC-EV) for test conditions. The plate was re-incubated on a Nikon BioStation CT under SCC to monitor wound closure by imaging at 20 min intervals for 24 h. The scratch area was measured using the BioStation IM/IM-Q Ver2.23 software.
For migration assays, transwell filters (0.8 µm pore size) (Corning, New York, NY, USA) were coated with fibronectin (1 mg/µL) (Sigma) prepared to a 10 µg/cm² coating density, then blocked with 0.1% FBS. OACs were added to the transwell at 2 × 10⁴ seeding density in 24-well plates containing DMEM, 0.1% FBS, and appropriate test conditions (negative control (0.1% FBS), positive control (10% FBS) or MSC-EV-treated (1:4 ratio OAC:MSC-EV)) and cultured for 24 h. transwell filters were swabbed and methanol fixed (Sigma). Cells were stained using the Diff-Quick kit (Fisher Scientific, Waltham, MA, USA) followed by ethanol dehydration (50%, 70%, 100%) and migrated cells were visualized using the EVOS XL Core cell imaging system (Life Technologies, Carlsbad, CA, USA).

Migration properties of GBM cells were analyzed in transwell (8 μm pore size; Corning Glass, Seoul, Korea). GBM cells (2 × 10⁴) were loaded in the upper well of the transwell and were incubated for 24 h. The migrated cells into the lower surface of the filter were then fixed and stained with a Diff Quick kit (Fisher, Pittsburgh, PA, USA). The number of migrated cells was counted in three microscopic fields per well.

For the invasion assay, the cells (2.5 × 10⁴ cells in serum-free medium) were seeded in the upper well of the Transwell chamber (8-mm pore size; Corning Glass, Corning, New York, NY, USA) that was precoated with 10 mg/mL growth factor-reduced Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). The lower well was filled with 0.8 mL of growth medium containing 10% fetal bovine serum as a chemoattractant. After incubation for 48 h at 37 °C, non-invaded cells on the upper surface of the filter were removed with a cotton swab, and migrated cells on the lower surface of the filter were fixed and stained with a Diff-Quick kit (Fisher, Waltham, MA, USA). Cells were imaged by phase contrast microscopy (Leica Microsystems, Wetzlar, Germany; magnification 10×). Invasiveness is determined by counting cells in five microscopic fields per well, and the extent of invasion is expressed as an average number of cells per microscopic field.