Oris 96 well cell migration assay kit

In vitro cell migration of hTCEpi cells was measured using the Oris^™ 96-well cell migration assay kit following the manufacturer’s instructions (Platypus Technologies, Madison, WI), as previously described (Makuloluwa and Shams, 2018 (link)). Briefly, hTCEpi cells were seeded in the wells of a plate around a stopper placed in the center of each well. The cells were incubated at 37 °C and cultured to full confluence surrounding the stopper. CH was then added at the above-described concentrations at the same time the stopper was removed to allow cells to migrate into the central detection zone for 12 h; Negative (vehicle) and positive controls (cytochalasin D, inhibit migration) were run simultaneously. Cells were fixed with 4% paraformaldehyde for 30 min and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) 1:5000. Cells were fluorescently imaged immediately after staining (Keyence Digital Microscope, Osaka, Japan). The area of the migration was measured using ImageJ software (ImageJ, US National Institutes of Health, Bethesda, MD) and the percentage of migration was calculated relative to the negative control.

HaCaT (ATCC, Manassas, VA), a spontaneously transformed immortal keratinocyte cell line from skin was maintained in high glucose DMEM medium (Gibco) supplemented with 10% FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin (Invitrogen), and TIGK (gift from Dr. Richard J. Lamont, University of Louisville), a telomerase immortalized epithelial cell line from oral mucosa (gingiva) was grown in DermaLife K medium (Lifeline Cell Technology) supplemented Life Factors (Lifeline Cell Technology), as previously described^{27 (link),44 (link)}. For functional analysis, miR-21 mimic and a control non-targeting miRNA mimic (GE Healthcare Dharmacon, Lafayette, CO), or anti-miR-10b LNA inhibitor and negative control LNA (QIAGEN, Hilden, Germany), were transfected into cells using DharmaFECT Transfection Reagent 1 as described previously^{45 (link)–47 (link)}. Cell proliferation was measured using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay as described previously^{26 (link),48 (link)}. In vitro cell migration was measured using the Oris 96-well cell migration assay kit (Platypus Technologies, Fitchburg, WI) as described previously^{45 (link)}. The cells that migrated into the detection zone were counted using ImageJ analysis software (version 1.421).

The cell migration was measured using the Oris™ 96-well cell migration assay kit (Platypus Technologies, Madison, USA) following the manufacturer’s instructions. Briefly, on the first day, 5 × 10⁴ cells were seeded in each well. On day 2, the stopper was removed to allow cells to migrate into the detection zone. Culture medium containing AG1478 (5 μM) or DMSO (control) was added and cell migration to central zone was monitored at 24 h and 48 h. The picture of the cells that migrated into the detection zone was taken using inverted microscope Leica DMI3000 B (Leica Microsystems, Germany).

The Oris™ 96-well cell migration assay kit (CMA1.101, Platypus Technologies) was used (3.5 × 10⁴ cells seeded/well). After plug removal, cells were treated without or with HGF (20 ng/ml) and PHA-665752 or ARQ197. Cell migration was monitored for 25 h using an automated ImageXpress Micro 2 (Molecular Devices) equipped with environmental control. Images were acquired at 5 h intervals with a 10× 0.2 NA Plan Apo objective (Nikon) and Roper CoolSNAP HQ camera (Roper Scientific). Wound closure was quantified using the threshold method in the MetaXpress software (Version MX 3.1.0.93).

Cell migration assay was performed using an Oris 96-well cell migration assay kit (Platypus Technologies, Madison, WI, USA) according to the manufacturer’s instructions as described previously44 (link). Briefly, 5 × 10⁴ cells were seeded in each well. After 1 h pretreatment with DMEM with 3% FBS containing RESV and recombinant PDGF-BB at 50 ng/ml with 5 μM aphidicolin (Sigma-Aldrich) to inhibit cell division, the stoppers were removed to allow cells to migrate into the detection zone. The cells were incubated for 48 h and stained with PBS containing calcein AM (Life Technologies) for 1 h. The area of cell migration was determined using Photoshop software (Adobe Systems, San Jose, CA, USA).