Incucyte flr microscope

To investigate the wound healing efficacy of a hydrogel comprising LMWP-GFs, QCN-NE, and OXY-PFOB-NE, an in vitro scratch-wound recovery assay was performed. A total of 2.5×10⁴ HaCaT or 4×10⁴ CCD-986sk cells (in 200 µL DMEM with 10% [v/v] FBS and 1% [w/v] penicillin/streptomycin) was added to each well of a collagen-coated Essen ImageLock 96-well plate (Essen Bioscience, Ann Arbor, MI, USA), and incubated at 37°C for 48 h to produce confluent monolayers. Cell-free scratches 700–800-µm wide were created in the monolayers using a 96-pin IncuCyte wound-maker (Essen BioScience), and the detached cells were washed twice with PBS (pH 7.4). The HaCaT and CCD-986sk cells were further treated with 200 µL of hydrogel diluted with DMEM containing 0.5% FBS to apply LMWP-GFs (LMWP-EGF [500 ng/mL], LMWP-IGF-I [500 ng/mL], LMWP-PDGF-A [10 ng/mL], and LMWP-bFGF [10 ng/mL]), QCN-NE (equivalent to 0.1 µg/mL of QCN), and 30 µg/mL of OXY-PFOB-NE alone or combined. The plates with the HaCaT and CCD-986sk cells were incubated at 37°C in an IncuCyte FLR microscope (Essen Bioscience) for 12 h and 48 h, respectively, and wound recovery was quantified using IncuCyte FLR image analysis software, which detects cell edges and generates an overlay mask to calculate relative wound density.

HeLa (cervical epitheloid carcinoma), MDA-MB-231 (mammary gland epithelial adenocarcinoma), A549 (alveolar basal epithelial adenocarcinoma) and RWPE-1 (HPV-18 immortalized prostate) cells, obtained from ATCC, were cultured in Eagle's minimal essential medium (EMEM), Dulbecco’s modified Eagle’s medium (DMEM) or Roswell Park Memorial Institute (RPMI) 1640 medium, with 10% fetal bovine serum and plated in replicate sets at 1,600 cells/well in 384-well plates 6-12 h prior to drugging. Cells were exposed for up to 72 h to 5-80 µM MINC1, DMSO (negative control) or 20 µM benzethonium chloride (positive cell killing control) to identify compounds that are directly toxic to cells. CellTox Green (Promega) was added to the growth medium according to manufacturer’s recommendations, and the cells were followed by live cell imaging using an IncuCyte FLR microscope (Essen Bioscience) to determine toxicity (CellTox Green fluorescence, Promega) and proliferation (phase contrast confluency). These image data were quantified with the IncuCyte application software (version 2011A rev2) and extracted cell toxicity and proliferation data were plotted with GraphPad Prism.

Next, an in vitro wound-healing assay was performed to compare the efficacy of inhibition of cancer cell proliferation/migration after the complex formation with DCK as well as incorporation into the nanoemulsion. The LLC or A549 cells were seeded at a density of 3×10⁴ cells/well in 200 μL of DMEM or RPMI medium containing 10% FBS on collagen-coated 96-well plates, respectively, and incubated at 37°C for 48 h to form a nearly confluent monolayer. Then, each well was carefully scratched to make a linear wound region (a cell-free zone) using a wound maker. The monolayer was washed twice with phosphate-buffered saline (PBS, pH 7.4) to remove the detached cells. Next, the cells were treated with free PMX, HP-beta-CD/PMX, HP-beta-CD/PMX/DCK, HP-beta-CD/PMX/DCK/P188, and HP-beta-CD/PMX/DCK/P188-NE in the respective media at concentrations equivalent to 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 μg/mL PMX and cultured at 37°C for 48 h. The drug-treated wells were photographed, and cell migration was monitored every 12 h by phase-contrast imaging with an IncuCyte FLR microscope (Essen Bioscience, Ann Arbor, MI, USA). IncuCyte FLR image analysis software was used to detect the cell edges automatically and to generate an overlay mask, which was used to calculate the cell inhibition area.