Incucyte live cell imaging system

LNCaP cells were seeded in a 96-well plate at 4000 cells per well, with up to 8 technical replicates per condition. After 24hrs, all cells were treated with NucView Caspase-3 Enzyme Substrate 488 (Biotium, 10402), and appropriate wells also treated with siRNA as described above, then placed in the Incucyte live cell imaging system (Sartorius). One image per well was taken every 2 h for 7 days. After 24 h in the Incucyte, the positive control cells were treated with either 50 µg/ml of TNFα (Sigma-Aldrich, H8916) + 100 nM of SM164 (Selleckchem, S7089), or 2 µM of Aphidicolin (Sigma-Aldrich, A0781). Integrated fluorescence intensity was calculated within the Incucyte Zoom software using the summed pixel intensity in calibrated units (CU) to determine the relative fluorescence units per image with the follow equation; CU x µm²/image. A significant change was calculated by one-way ANOVA, with each BRG1 on-target siRNA compared to the non-targeting control. The results were corrected for multiple comparisons with Dunnett’s test. A significant change is defined as ****p < 0.0001.

Live-cell tracking of A549 cells expressing the mVenus-Gem1 reporter was carried out using the IncuCyte live-cell imaging system (Sartorius). The cells were plated at a density of 40,000 cells per well on six-well plates, and the 4-day long treatments were started 24 h after plating. Imaging was performed every 45 min using 10× objective and 150 ms exposure time for the FITC channel. Cell tracking was carried out manually using the IncuCyte software. Only cells that were mVenus-Gem1 positive at some point during the 24-h period before chemical treatment were tracked. Manual tracking recorded timings of cell cycle state change and/or cell death, as detected on the basis of cell morphology. The tracking of each cell lineage lasted until the division of second cell generation or until the end of the 4-day drug treatment. If a cell remained arrested in a specific cell cycle state until the conclusion of the experiment, the duration of that cell cycle state was calculated to end at the conclusion of the experiment. Consequently, for a small fraction of cells, the durations of some cell cycle phases are underestimated.

CHO-K1 cells transfected with EGFP, Y2R-EGFP, and Y5R-EGFP were seeded in IncuCyte® ImageLock 96-well plates at a density of 2–2.5 × 10⁵ cells per well. Eighteen hours after seeding, a scratch was made in the confluent monolayer using the 96-well Wound Maker™. Cells were then washed with serum-free medium to clear any floating cells within the scratch prior to treatment. The subsequent migration monitoring was performed in media with three different serum concentrations, 10%, 1%, or 0.1% FBS, depending on the experimental design. For the low-serum conditions, cells were primed in serum-free media for 6 h before creating the scratch and treating with media supplemented with 1% or 0.1% FBS and NPY or its receptor antagonists, when desired. Subsequently, the 96-well-plates were placed in the IncuCyte live cell imaging system (Sartorius, Goettingen, Germany) and the images collected every 2 h during the incubation time. The IncuCyte ZOOM software generated a wound width (WW) migration metric, which was used to calculate the migration distance (MD) according to the following formula: MD = (WW_T0 – WW_Tn)/2.