Ckx31 11 php

Cell migration was assessed by performing a wound healing assay, as previously described (29 (link)). Briefly, the cells were cultured in 6-well plates (5×10⁵ cells/well). When the cells reached 90% confluence, a single wound was created in the center of the cell monolayer using a P-200 pipette tip. At 0 and 24 h following incubation at 37°C, wound closure areas were visualized using a phase-contrast microscope (CKX31-11 PHP; Olympus) at ×100 magnification.

The number of live cells in EpH4/3T3L1 cells aggregates without or with FN-treated GM was determined by counting the number of cells nuclei after their crystal violet staining [41] (link). Briefly, a mixed solution of 0.2 M citric acid and 0.2 wt % crystal violet was added (100 μl/well) to each well of 96-multiwell culture plate 7 days after EpH4/3T3L1 cells were cultured without or with FN-treated GM dehydrothermally crosslinked for 48 at 140 °C. After the crystal violet staining, the cells were lysed in 0.1 wt % Triton X-100 in PBS at 37 °C overnight to separate the nuclei from the cell debris. After pipetting, the cell nuclei collected were viewed on a light microscope (CKX31-11PHP, Olympus, Tokyo, Japan) and counted in a hemocytometer (OneCell Inc., Hiroshima, Japan). The nuclei of live cells are generally round, while that of dead cells are irregularly shaped. Live cells were distinguished from dead cells by their nucleus shape.

Invasion assays were performed using 48-well Boyden chambers (Neuroprobe, Gaithersburg, MD, USA). The lower wells of the chamber were filled with a standard culture medium. The chamber was assembled using polycarbonate filters (Neuroprobe) coated with Matrigel. Cells in a serum-free medium (5×10⁴ cells/well) were seeded in the upper compartment of the chamber. After incubation for 24 h, cell migration was quantified by counting the number of migrated cells after staining with hematoxylin and eosin under a light microscope (Olympus, CKX31-11 PHP, Tokyo, Japan). The data shown are the mean values of three independent experiments.

The number of live cells in EpH4 cell aggregates with MM, GM or matrigel-coated GM incorporation was determined by counting the number of cell nuclei after the crystal violet staining [28] (link). Briefly, a mixed solution of 0.2 M citric acid and 0.2 wt% crystal violet was added (100 μl/well) to each well of well plate 7 days after EpH4 cell culture with MM, GM, and matrigel-coated GM. After crystal violet staining the cells were lysed in 0.1 wt% Triton X-100 in PBS at 37 °C overnight to extract the nuclei from the cells. After pipetting, the nuclei collected were viewed under a microscope (CKX31-11PHP, Olympus Ltd, Tokyo, Japan) and counted in a hemocytometer (OneCell Inc., Hiroshima, Japan). The nuclei of live cells are generally round, while dead cell nuclei are irregularly shaped. Base on the nucleus shape, live cells can be distinguished from dead cells to assess the number of live cells.