Incucyte imagelock 96 well plate

PDAC cells transduced with doxycycline-inducible pCW-CEBPD (and mVenus fluorophore) or pCW-CTRL (and mCherry fluorophore) constructs were mixed at equal ratios and seeded at 20,000 cells per well in the Incucyte Imagelock 96-well plate (#BA-04856, Sartorius, Göttingen, Germany) and allowed to attach overnight in complete medium. Doxycycline was added to half of the wells at a concentration of 2 µg/mL. The next day, cell layers were wounded using the Incucyte wound maker tool (#4563 and #5025-0191, Sartorius), the cells were washed once with PBS, and fresh complete medium with 1.5–2.5% FCS and with or without doxycycline was added and refreshed every 2–3 days. Cells were imaged at regular intervals using the Incucyte S3 Live-Cell Analysis Instrument (Sartorius). The Incucyte Scratch Wound Analysis Software Module (#9600-0012, Sartorius) was used to quantify the percentage of initial wound area covered by red fluorescent or green fluorescent cells. To correct for differences in baseline migration between CTRL and C/EBPδ-inducible cells, the following formula was used: C/EBPδ_{doxycycline(corrected)} = C/EBPδ_doxycycline ∗ (CTRL_untreated/C/EBPδ_untreated). Graphs and linear regression to compare migration slopes were done using GraphPad Prism (version 9.1.0, GraphPad Software Inc.).

Smooth muscle cells isolated from mouse aorta were subjected to proliferation and migration assays as previously described [23 (link)]. Briefly, experiments were performed using the IncuCyte S3 Live-Cell Analysis System (Sartorius). For the proliferation assay, cells were plated at 60–70% confluence on a regular 48-well plate, and images were taken every 2 h for 96 h. For the migration assay (wound-healing assay), cells were plated at 100% confluence on an IncuCyte ImageLock 96-well plate (Sartorius), and a homogeneous 700–800 μm wide wound was created using the IncuCyte WoundMaker tool (Sartorius). Images were taken every 2 h for 60 h. For both assays, images were analyzed using IncuCyte Base software with the Scratch Wound Analysis Software Module (Sartorius). Experiments were repeated with smooth muscle cells originating from 3 independent isolations for each genotype.

OVC316 cells (25 × 10⁵) were seeded into an Incucyte® Imagelock 96-well plate (Essen Bioscience, 4379, Hertfordshire, UK) in medium with 10% FBS. Cells were wounded after 48 h incubation with Incucyte® 96-well WoundMaker Tool (Sartorius, 4563, Goettingen, Germany). Each well was washed with 200 µl PBS, after which 200 µl of the culture medium with indicated drugs were added. The plate was pre-incubated 30 min and imaged as indicated. The images were analyzed using the Incucyte® ZOOM software.

6·10⁴ cells were seeded on Incucyte ImageLock 96-well Plate (Essen BioScience Inc. 4379) and cultured for 24–48 h to reach full confluency. The wound was done using the Woundmaker 96 tool (Essen Bioscience Inc.) and the migration of the cells to heal the scratch was analyzed using IncuCyte S3 Live-Cell Analysis System (Essen Bioscience Inc.).

HaCaT cells (7 × 10⁴ cells per well) were seeded in an Incucyte^® ImageLock 96-well plate (Essen BioScience, Ann Arbor, MI, USA) and grown in DMEM (Anprotec, Bruckberg, Germany) supplemented with 10% FBS (PAN-Biotech, Aidenbach, Germany) and 1% Zellshield (Biochrom, Berlin, Germany). The following day, 10 µg/mL mitomycin C (Merck, Darmstadt, Germany) was added, and after 3 h, the medium was changed to DMEM supplemented with 0.5% FBS. A 700–800 µm wide scratch was applied in the cell-monolayer with IncuCyte^® WoundMaker (Essen BioScience, Ann Arbor, MI, USA), and the remaining cells were washed twice with DPBS (Anprotec, Bruckberg, Germany). Recombinant CXCL12 wt and its variants in DMEM with 0.5% FBS (100 nM final protein concentration) were added to the cells. The cells were incubated for up to five days, and migration was determined as closed part of initial wound area every 2 h with IncuCyte^® scratch wound cell migration software module (Essen BioScience, Ann Arbor, MI, USA).

For wound healing assays, fibroblasts were plated in the wells of an Incucyte™ ImageLock™ 96-well plate (Essen BioScience) and the WoundMaker™ tool was used to create a denuded area in each well on the plate. The IncuCyte™ ZOOM live-cell analysis system (Essen BioScience) was used to automatically collect time-lapse images (phase-contrast) and to quantify cell migration over time as the density of cells in the denuded area relative to the density of cells out of the denuded area (relative wound density). Plots were determined to be statistically significantly different based on repeated measures two-way ANOVA with Dunnett’s multiple comparison test.