Dm il led microscope

ShCTRL, sh599, and sh1552 OVCAR3, IGROV1, SKOV3 and MES-OV cell migratory capacity was determined in vitro by both wound healing and transwell migration assays, as previously described (28 (link)).
For wound healing assay, confluent cells were scratched with a p200 pipet tip. Wells were washed to remove detached cells, and medium was replaced with serum-free RPMI-1640 or DMEM (for MES-OV cells). At time 0 and after 24 and 48 h, pictures of the wounded area were taken with Leica DM IL LED microscope (Wetzlar, Germany). The distance between scratch edges was quantified using ImageJ software.
For transwell migration assay, 5 × 10⁴ cells resuspended in 200 μL of RPMI-1640 supplemented with 0.2% FBS were seeded into 8 μm pore cell culture insert (migration chambers, Falcon, Corning) in 24-well plates. Wells were filled with 800 μL of RPMI-1640 medium containing 20% FBS, and cells were incubated at 37°C. After 18 h, cells that had not crossed the membrane were removed with a cotton swab, and inserts were fixed with 4% PFA. Cells on the bottom of the membrane were stained with crystal violet. Images of five fields per insert were taken with a Leica DM IL LED microscope and the area covered by migrated cells was quantified using ImageJ software.

βTC6 cells and MS1 cells were co-cultured using Falcon® Cell culture inserts and their companion plates (Corning Inc., NY, USA). βTC6 cells were grown in Falcon® Permeable Support and MS1 cells were seeded in the lower chamber of the plate. The number of MS1 cells was determined by cell counting with a hemocytometer under Leica DM IL LED microscope (Leica Microsystems Ltd, Wetzlar, Germany). For wound healing assay, the scratch was made in monolayer culture of MS1 cells using a 200-μL pipette tip, and the wound closure was analyzed using a Leica DM IL LED microscope (Leica Microsystems Ltd) 24 h later.

For mineralization assays, 5 × 10⁴ ECs were seeded into 48‐well plates and incubated in osteogenic medium containing 10⁻⁸M/L dexamethasone, 0.2mM/L ascorbic acid, and 10mM/L β‐glycerolphosphate in the presence of BMP/ TGF‐β ligands for 28 days. The medium was refreshed every 4 days. Afterwards, cells were washed twice with PBS and fixed with 3.7% formaldehyde for 5 min. Next, cells were washed twice with distilled water; measurement of calcium deposition was performed by Alizarin Red solution (ARS) staining, as previously described.16 Precipitates, originated from three independent ARS assays, were dissolved using 10% cetylpyridinium chloride, and absorbance was measured at 570 nm. Representative pictures were obtained using a Leica DMIL LED microscope (Leica, Wetzlar, Germany) with 10× magnification.