Live dead cell assay kit

HaCaT cells treated with PM₁₀ and tart cherry were labeled with a live/dead cell assay kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s protocol. Live cells with esterase activity (green) and dead cells compromising plasma membranes (red) were directly counted per random microscopic field and the numbers were converted to a percentage by multiplying by 100. Ten random fields per coverslip were counted. Nuclear translocalization of NF-κB was determined by immunofluorescence staining as previously described [12 (link)]. Cells incubated with phospho-NF-κB antibody were labeled with goat anti-rabbit IgG/IgM (H + L) (Invitrogen Co., Carlsbad, CA, USA) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) in 5% normal goat serum. The immunofluorescence signals were detected using an Olympus FluoView™ 300 confocal microscope (Center Valley, PA, USA) with 400× objective.

U87-MG cells were seeded onto coverslips in 24 well plates at 5 × 10⁴ cells per well, and then treated with 200 nM of FL3 for 48 h. After 48 h, the treatment was stopped, and the wells were washed with PBS. The cells were trypsinized and then centrifuged to have a unique pellet. The cell pellet was resuspended in the live/dead assay dye solution for 10 min (Live/dead cell assay kit, Abcam, ab115347), and then visualized under an epifluorescence microscope (Leica DM4000 B).

To investigate cell viability after BES with PEI/DNA, a Live/Dead Cell Assay kit (Abcam ab115347, Mitosciences, USA) was used after 1 day of bio-electrospray with a branched 25 kD PEI/DNA complex. When bio-electrospray was run on the dish, the density of cells was 5 × 10⁵ mL⁻¹ and the electrospray voltage was varied (0 kV, 10 kV, 15 kV, and 20 kV). The concentration of provided live/dead dye was 1000×. When dye was used for the assay, it had to be diluted 5× in PBS. The cells were stained with 5× live/dead dye and incubated 20 minutes at room temperature. Then, the dyed cells were observed by fluorescence microscopy (Ti-E, Nikon, Japan).