Nucview 488 caspase 3 assay kit for live cells

To assess the activity of engineered suicide genes, StaPLd-Casp9-expressing or iCasp9-expressing HeLa cells were stained after drug incubation. Media were collected to harvest dead/lifted cells, adherent cells were trypsinized to harvest living cells, and the two were pooled and pelleted at 500g for 5 min. Cells were washed once with HBSS, resuspended in either HBSS or Annexin V Binding Buffer (Biotium), and stained with the NucView 488 Caspase-3 Assay Kit for Live Cells (Biotium) or the Annexin V CF488A Conjugate kit (Biotium), respectively, according to manufacturer’s instructions. In some instances, cells of the parent cell line (Flp-In HeLa) were annexin V-stained in parallel. For medium-term preservation, cells were fixed in 4% PFA in PBS for 15 min at ambient temperature following staining, centrifuged at 10,000g, and resuspended in HBSS after aspirating the PFA. For microscopy, a 25 μL droplet of each sample was then placed on a Superfrost Plus glass slide (Fisher Scientific), and allowed to dry partially, before adding Vectashield Mounting Medium with DAPI (Vector Labs) and a #1.5 cover glass (Fisher Scientific). Slides were sealed with clear nail polish and kept at 4 °C.

Caspase-3 activity was detected using NucView 488 Caspase-3 Assay Kit for Live Cells (Biotium, Catalog No. 30029). Briefly, A375 and A375-R cells were seeded in 10 cm plates, treated with vehicle or compound for 72 h, and then the cells were trypsinized and resuspended at a density of 1 × 10⁶ cells/mL. Cell suspension (0.2 mL) was pipetted into a test tube and incubated with a 5 µM NucView 488 substrate concentration for 30 min at room temperature in the dark. After that, 300 µL of PBS was added into each test tube and analyzed by flow cytometry. The intensity of the fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 515 nm.