Live dead viability cytotoxicity kit

NIH3T3 cells were cultured in DMEM with 10% FBS and 1% antibiotics at 5% CO₂ with humidity at 37 °C. For the culture of HeLa cells, MEM was used instead of DMEM. Prior to cell seeding, the pigmented A4 paper was washed with 70% ethanol in water and air-dried for sterilization. For biocompatibility test, NIH3T3 cells (6 × 10⁴ cells) were seeded on each well of an 8-well plate containing 0.7 cm² paper and cultured for 24 h in a cell culture incubator under 5% CO₂ with humidity at 37 °C. Subsequently, cells on the paper were stained with ethidium homodimer-1 and calcein-AM following the manufacturer’s guidelines for the commercial LIVE/DEAD^TM viability/cytotoxicity kit from Invitrogen^TM. For the photothermal ablation test, HeLa cells (5 × 10⁴ cells) were seeded on each well of an 8-well plate containing a 0.7 cm² sized paper having both bare and pigmented regions together. After 24 h of culture, cells on the paper were washed with 1× PBS and irradiated with NIR light (808 nm, 1 W/cm²) in 1× PBS for 10 min and incubated for 5 h in cell culture media. Finally, the viability of cells remaining on the paper was evaluated by the aforementioned LIVE/DEAD^TM viability/cytotoxicity kit from Invitrogen^TM. Images were analyzed using ImageJ software to quantify both the live and dead cells attached to the surface.

Endometrial cell viability was evaluated using a LiveDead Viability/Cytotoxicity kit (Life Technologies) according to manufacturer’s instructions. Briefly, 3-D endometrial cell structures were washed with PBS, incubated with 2 μM calcein AM and 4 μM Ethidium homodimer-1 (EthD-1) in PBS for 45 minutes at room temperature in the dark, washed twice, mounted directly in the dish under coverslips with Vectashield (Vector Laboratories), sealed with nail polish, and observed at 518 nm and 593 nm with an Olympus BX41 microscope equipped with epifluorescence. Three photos were taken per dish at 200X magnification. The percentage of live cells were calculated by dividing the number of live cells by the total number of cells counted with the Multi-point tool in ImageJ (National Institutes of Health).

The following compounds and reagents were used in this investigation: DTG (Tocris Biosciences, Ellisville, MO, United States); Live/Dead Viability/Cytotoxicity Kit (Life Technologies); Sodium Azide (ACROS Organics, Fair Lawn, NJ, United States); Fura-2AM (Molecular Probes, Eugene, OR, United States). The following antibodies were also used: Alexa Fluor 488 anti-mouse IgG (A-11001) and anti-rabbit IgG (A-11008) (Life Technologies); anti-Bax (ab5714), anti-activated caspase-3 (ab32351) and anti-Bcl-2 (ab32370) (Abcam, Cambridge, MA, United States). Afobazole was generously provided by IBC Generium (Moscow, Russia). The vehicle for drugs and reagents used were either water (H₂O), ethanol (EtOH) or dimethyl sulfoxide (DMSO), and appropriate vehicle controls were carried out for each study.

Viability assay was performed on DFAT cells in cell fibers 7 and 21 days after differentiation induction using LIVE/DEAD Viability/Cytotoxicity kit (Life Technologies, Carlsbad, CA, USA). DFAT cell fibers from both the control and differentiation induction groups were incubated in live/dead viability assay working solution (1 μM calcein AM + 1 μM ethidium homodimer-1 in PBS (+)) for 30 minutes at 37°C. DFAT cell fibers were subsequently imaged by phase contrast and fluorescence microscopy (IX71, Olympus, Tokyo, Japan).

Primary adult rat cardiac fibroblasts were grown in Permanox plastic chamber slides (Nunc, Thermo Fisher Scientific) in DME/F12 medium (HyClone, GE Healthcare) supplemented with 0.5 % FBS and 500 μmol/L ascorbic acid. Cells were infected with AdshLacZ (control) or AdshScx adenovirus at MOI 200 for 48 h. Cell viability was assayed using the LIVE/DEAD Viability/Cytotoxicity kit as per manufacturer’s instructions (Life Technologies). Vital dyes calcein acetoxymethyl ester (calcein AM) and ethidium homodimer-1 were used to visualize live (green) and dead (red) cells, respectively, using a Zeiss Axio Imager M1 epifluorescence microscope with 10× EC Plan-Neofluar objective (NA 0.3). Data was recorded as percent cell death compared to control from three independent experiments (minimum total cell count of 400); 200 μmol/L H₂O₂-treated cells were used as dead cell control.