Incucyte caspase 3 7 green apoptosis assay reagent

LN443-Cas9 cells were transduced with guide RNA sgCtrl or sgVRK1. Following 1 week under selection, 5 × 10⁴ cells per well were seeded in 24-well Falcon plates (Thermo Fisher Scientific, catalog 353047). Then 5 mM of IncuCyte Caspase-3/7 Green Apoptosis Assay Reagent (Sartorius, catalog 4440) as well as 1:500 of Nuclight Rapid Red Dye (Sartorius, catalog 4717) were added to each well. The plate was transferred into the IncuCyte S3 Live-Cell Analysis System (Sartorius, catalog 4647) for imaging. Phase contrast images and green/red fluorescence channel images were captured using the 10× objective magnification every 4 hours for a total of 48 hours. For each well, 4 images containing both phase contrast and green channel data were obtained.
Using the IncuCyte S3 Analysis System software, cell confluence over time was quantified along with the intensity of green (apoptosis-positive) objects in mm²/well. Computer-generated masks for confluence and green area, trained on a sample set of images across time points and confluence levels, were manually checked for accuracy. Each metric was averaged over the 4 quadrants per well. First, the green object total intensity metric for each well was divided by the confluence metric for each well, yielding a normalized measure of caspase-3/7 activity.

Human HS11, HS01, HS02, HS13, and HS05 and mouse MTC-10 cells (1000 cells/well) were seeded in growth medium in three replicate wells/condition of a 384-well CellBIND Corning plate. After ~6 h, drugs (diluted in 0.001% DMSO) at three increasing concentrations (EC50 as well as lower and higher concentrations) and Incucyte® Caspase-3/7 Green Apoptosis Assay Reagent (Sartorius, Gottingen, Germany) were added to the wells. Wells were imaged using the Incucyte® S5 Live-Cell Analysis system (Sartorius) for 24–72 h. Phase and green fluorescent images were collected every 2 h and analyzed using the integrated basic analyzer software.

The activation of caspases was measured by long-term video-microscopy. The cells were transfected as described above and/or treated with Carboplatin. 1 h after treatment IncuCyte Caspase-3/7 Green Apoptosis Assay Reagent or IncuCyte AnnexinV Red Reagent (Sartorius, Goettingen, Germany) was added to a final concentration of 5 nM as well as PI at a final concentration of 10 μg/ml (Sigma Aldrich)^{52 (link)}. The cells were placed into the IncuCyte ZOOM Live-Cell Analysis System detecting red and green florescence. The imaging system is placed in an incubator with standard culture conditions. Pictures from each well in two different positions were taken automatically every hour with a 20x objective and analysed with the IncuCyte ZOOM Software for the amount of fluorescent stained cells. The data were visualized using GraphPad Prism 5 software.

Human HS11, HS01, HS02, HS13 and HS05 and mouse MTC-10 cells (1,000 cells/well) were seeded in growth medium in three replicate wells/condition of a 384-well CellBIND Corning plate. After ~ 6h, drugs (diluted in 0.001% DMSO) at three increasing concentrations (EC50 as well as lower and higher concentrations) and Incucyte^® Caspase-3/7 Green Apoptosis Assay Reagent (Sartorius, Gottingen, Germany) were added to the wells. Wells were imaged using the Incucyte^® S5 Live-Cell Analysis system (Sartorius) for 24–72h. Phase and green fluorescent images were collected every 2h and analyzed using the integrated basic analyzer software.

Cells were seeded with IncuCyte^® Caspase‐3/7 green apoptosis assay reagent (Sartorius, Royston, UK; #4440). Plates were imaged in an IncuCyte Zoom (Sartorius, Royston, UK) acquiring images every 3 h over a 72‐h period using the ‘phase’ and ‘green’ channels. Images were analysed using the IncuCyte Zoom software (Sartorius).

Cell lines were transduced with lentivirus as described above to stably express nuclear-localized mKate2, enabling live cell imaging. Cells were plated at a density of 4–5 $\times$ 10³ cells per well in 96-well tissue culture plates (CoStar), with a minimum of 5 replicate wells per experimental condition. Following a 24 h incubation period, the media was replaced with media containing IncuCyte® Caspase-3/7 Green Apoptosis Assay Reagent (final concentration 5 µM, Sartorius, #4440). Concurrently, cells were treated with either vehicle (DMSO) or CDDD11-8. Plates were imaged on the Sartorius IncuCyte S3 Live Cell Analysis System (RRID:SCR_023147) for 5 d, capturing images in the red and green channels using a 10 $\times$ objective. Resultant images were analysed to determine the number of live (red; mKate2) and dead (green; Caspase-3/7) cells using the associated IncuCyte S3 software. Image analysis was trained using six representative images from both low and high confluence, vehicle and CDDD11-8 treated cells. Three independent proliferation assays were conducted for each cell line to determine a robust half-maximal inhibitory concentration (IC₅₀).