Incucyte caspase 3 7 green dye for apoptosis

A black-walled 96-well plate (#6005182, PerkinElmer, Waltham, MA, United States) was coated with 50 µl myogel (0.5 mg/ml) per well. The next day, HSC-3 and UT-SCC-24A cells were stained with CellTrace Far Red (Invitrogen, Carlsbad, CA, USA) by resuspending 500,000 cells in 500 µl PBS and adding 0.5 µl of the dye before incubating the cells at 37°C for 20 min. Next, 2.5 ml of complete media was added to the cells, and they were incubated for another 5 min. The cells were then centrifuged and resuspended in complete media. The media remaining in the wells of the 96-well plate after myogel coating was removed and the cells were seeded with 1,000 cells/well in 100 µl of media. The cells were left to adhere overnight, and the following day, the media was replaced with 100 µl of media containing the Incucyte® caspase-3/7 green dye for apoptosis (Sartorius, Göttingen, Germany) (diluted 1:1,000), as well as 5 or 10 µM of the MPMs or 10 µM cisplatin. Control wells only contained media and the caspase-3/7 green dye. The plate was placed in the Incucyte S3 (Sartorius) and imaged at ×10 magnification every 2 hours for 24 h in total. Each condition had triplicate wells, and four images were taken per well. The experiment was conducted three times. The Incucyte software was used to analyze the area of red fluorescence (proliferation) and number of green and red objects (apoptotic cells).

Cells plated in 96 MW and treated as indicated were monitored overtime using Incucyte S3 or S5 live-cell analysis system (Sartorius). For detection of apoptotic cells, Incucyte caspase 3/7 green dye for apoptosis (Sartorius) was added 1:1000 to cells one day after ASO transfection, to prevent interaction between ASO and dye. Growth rate (GR) was calculated as reported in ref. ^{63 (link)}. For IC₅₀ calculation, values were computed using Prism software.