Inverted phase microscope

The Transwell invasion assay was performed using 24-well Transwell plates with polycarbonate membrane and 8.0 μm pores (Corning, USA). After 24 h of transfection, Matrigel (4.0 μg/μl, 60 μl) was added into Transwell chambers (Corning, NY, USA) and cultured for 2–3 h at 37°C for gel solidification. RPMI 1640 medium containing 10% FBS was added to the lower chambers and cells were seeded in the upper chambers with serum-free medium at a density of 4 × 10³ cells/per well. After 48 h, the migrated cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 30 min. Migrating cell numbers were counted in five random fields under an inverted phase microscope (Nikon, Japan) at a magnification of 100× as previously described [13 (link)]. Assays were performed in triplicate followed by statistical analysis.

The chondrocytes were examined under a Leica DM4000B microscope (Leica, IL, USA) for any cellular morphological changes in the presence of the control (DMEM), 10 ng/mL IL-1β alone, or in combination with different concentrations (1 nM, 10 nM, 100 nM, and 1 µM) and times (0, 24, 48, and 96 h) of ZOL (Aladin, Shanghai, China). IL-1β-induced IMCCs were treated with 10 or 100 nM ZOL for 24 or 48 h. Caspase-9 (apoptosis initiator) and caspase-3 (apoptosis effector) were examined by qRT-PCR and western blotting. The percentage of apoptotic cells was determined by flow cytometry. The expression of the proliferating cell nuclear antigen (PCNA) was determined [29 (link)]. XAV-939 (Selleck Chemicals, TX, USA) and LiCl (Sigma-Aldrich Co., MO, USA) were used as the inhibitor and activator of the Wnt/β-catenin signaling pathway, respectively, and were added to the ZOL-treated TMJOA chondrocytes for 24 and 48 h. Chondrocytes without ZOL were used as controls. The changes in chondrocyte morphology were examined using an inverted phase microscope (Nikon, Tokyo, Japan).

Cultures were examined with a Nikon inverted-phase microscope at × 40. Colony counts were made 15 days after plating. Aggregates of 50 or more cells were considered colonies. Aggregates of less than 50 cells were considered clusters. The plates were placed under an inverted microscope, and 10 visual fields were chosen at random, and the number of colonies greater than 50 cell or 0.05 mm in field of vision were counted.

A scratch wound assay was used to evaluate cell migration as previously described. First of all, fibroblasts were seeded in 6-well plates with and without co-culture of microfat. When reached confluence, cells were dealt with serum depleted medium for another 12 h. A scratch wound was then made in the middle of each well by 200-µL pipette tips. After washing three times with PBS, the gap of the scratch was recorded at 0 and 24 h by an inverted phase microscope (Nikon, Japan). Finally, using the Image-Pro Plus system to analyze the wound area. Three independent fields were analyzed in each well, and each experiment was performed in triplicate.

Wound healing assay reflects the migration behavior of HSFs. First of all, HSFs were seeded in 6-well plates with complete medium. When reached confluence, cells were dealt with serum-depleted medium for another 12 h. A scratch wound was then made in the middle of each well by 1 ml pipette tips. After washing three times with phosphate buffer saline (PBS), HSFs were treated with HKL at 0 μg/ml or 6 μg/ml for 1 day. The gap of scratch was recorded at 0, 6, 12, 18, and 24 by inverted phase microscope (Nikon, Japan). Finally, using Image-Pro Plus system to analyze the wound area.