Essen imagelock

For kinetic migration analysis, cells were seeded to ~90% confluency in 96-well plates (Essen ImageLock, Essen Biosciences, Ann Arbor, MI, USA). Wound areas were made with a 96-pin Wound Maker device and washed with PBS to prevent reattachment of dislodged cells. Cells were treated with salinomycin immediately after wound scratching, and images of the scratched fields were automatically acquired and registered every hour for 24 h with an IncuCyte^™ ZOOM^® Kinetic Imaging System. The relative wound density was analyzed using the IncuCyte^™ Scratch Wound Cell Migration Software Module.

Approximately 5 × 10⁴ EA.hy926 cells/well were seeded in a 96-well microplate Essen ImageLock™ (4379, Essen Bioscience, Ann Arbor, MI, USA) and cultured overnight. Then homogeneous 700–800 micron-wide scratch wounds were made using a WoundMaker™ device (4563, Essen Bioscience, Ann Arbor, MI, USA) containing 96 pins. The compounds and/or ligands were added to each well in 100 µL of fresh serum-free medium. Each group contained 6 replicates. Real-time pictures were acquired every 4 h by the IncuCyte ZOOM (Essen Bioscience) to track the wound closure. The wound density was analyzed using the software package provided by the manufacturer and normalized to the wound width at the start of the experiment. All the experiments were repeated at least three times, and representative results are shown.

Cells were seeded in gelatin coated 96-well plates (Essen ImageLock, Essen Bioscience) at 80% confluence and incubated overnight to allow them to reach confluence. Cells were treated for 2 hr before wounding with 2 μg/ml mitomycin C (M4287, Sigma) to block cell proliferation and scratches were performed in the cell monolayer using the wound maker (Essen Bioscience). Cells were washed immediately three times with PBS and re-fed with growth medium. Cell migration was monitored with an Incucyte FLR Live-Cell imaging system equipped with a 20X objective (Essen Bioscience). Images were acquired every 3 hr for 48 hr. Migration was quantified by measuring the relative wound density of at least three biological replicates in each experiment, using the Incucyte software (Essen Bioscience) as recommended by the manufacturer.

U251 cells (750cell/well) were transfected with miRNA mimics at 25 nM and grown in a 96-well Essen ImageLock cell culture plate (Essen BioScience). Percentage confluence was monitored using the high definition automated imaging system from IncuCyte (Essen BioScience) following the manufacturer's direction. The experiments were performed in quadruplicate. The data was analyzed using analysis of variance and displayed as mean ± standard deviation.

To distinguish CT26 from MSCs, green fluorescent protein (GFP) and puromycin resistance genes were transfected into CT26 colon cancer cells using copGFP control lentiviral particles (sc-108,084; Santa Cruz Biotechnology (Santa Cruz, CA, USA)) according to the manufacturer’s protocol as previously described [21 (link)]. The cells were maintained in a complete medium containing puromycin for two weeks after transduction. In the co-culture experiment, CT26-GFP and MSCs were maintained in RPMI1640 with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), a penicillin–streptomycin mixture, and 1 ng/mL of fibroblast growth factor-2. CT26-GFP cells (cell density, 3 × 10⁴ cells per well) cultured with MSCs (cell density, 3 × 10⁴ cells per well) were seeded into 24-well plates (Essen ImageLock; Essen Bioscience, Ann Arbor, MI, USA). The cells were treated with JPH203 at a 1 μM concentration. The proliferation of CT26-GFP cells and MSCs was measured using a label-free, high-content time-lapse assay system (Incucyte Zoom; Essen BioScience, Ann Arbor, MI, USA). CT26 and MSCs proliferation abilities were calculated separately according to the difference in color.