Live dead cell staining kit

Cell viability was determined 24 h after stimulation by live/dead staining using the Live-Dead Cell Staining Kit from Enzo (Enzo, Framingdale, NY, USA). After washing the adherent cells in cell culture plates with 1xPBS (phosphate buffered saline) three times, 1 mL staining solution (1 µL solution A + 1 µL solution B in 1 mL staining buffer) were added per well. After the incubation at 37 °C for 15 min, the fluorescence of green fluorescent Live-Dye™ (Ex/Em = 488/518 nm) and propidium iodide (Ex/Em = 488/515 nm) were determined by ECHO Revolve fluorescence microscope (Echo, San Diego, CA, USA) using four-fold magnification and an exposure time of 670 ms for both fluorescence dyes.

The live–dead analysis was performed after 24 h, 72 h, and 168 h of incubation using the Live/Dead Cell Staining Kit from Enzo Life Sciences (Lausen, Switzerland) following the manufacturer’s guidelines. Briefly, after washing the cells with 1 × PBS, 150 µL of staining solution containing calcein-AM and propidium iodide for staining live and dead cells, respectively, was added per well followed by incubation for 15 min at 37 °C, 5% CO₂, and 95% humidity. Afterwards, the fluorescence staining was directly visualized using the EchoRevolve fluorescence microscope (Echo, San Diego, CA, USA), distinguishing between calcein-AM (green dye, Ex/Em = 488/515 nm) and propidium iodide (Ex/Em = 570/602 nm).

The pumped artificial vessel was soaked with a Live/Dead cell staining kit (Enzo Life Sciences, NY, USA), composed of Live-Dye™ and propidium iodide (PI), based on the permeability of cell membrane integrity. Microscopic images were obtained at a random area with a fluorescence image using the Olympus DP71 microscope digital camera installed on the Olympus BX51TF system microscope (Olympus, Tokyo, Japan). The image was then merged with the color channel merge method using the ImageJ software (National Institute of Health, NY, USA).

GF and PDLF cultures were stained with Live-Dead Cell Staining Kit (Enzo Life Sciences AG, Lausen, TX, USA) according to the instructions by the manufacturer. Cultures were evaluated using fluorescence microscopy for green and red dyes, with a B-2A filter (excitation filter wavelengths, 450–490 nm). Vital cells appeared green, while dead cells appeared red in indirect cell cultures. Images were taken at 100-fold magnification. Unexposed 2D cell cultures and those treated with staurosporine served as a positive and negative control, respectively.

Cell proliferation/viability was measured using a cell counting kit (CCK‐8, Dojindo Laboratories, Japan) as previously described (Andrukhov et al., 2015). In these experiments, 1 × 10⁴ cells were seeded on either BHM or MCM (the porous layer facing up) membrane in 200 μl DMEM. Cells seeded at the same density on tissue culture plastic (TCP) were used as control. Cell proliferation/viability was measured after 3, 7, and 14 days of culture. Then, 20 μl of CCK‐8 reagent was added into each well and the culture plates were incubated in 5% CO₂ at 37°C for 2 hr. Thereafter, 100 μl of each culture solution was transferred to a separate 96‐well plate and the optical density (OD) was measured at 450 nm using a microplate reader (Synergy HTX; BioTek) at 450 nm. The experiments were repeated five times with the cells isolated from five different donors.
Cell viability was further visualized using the LIVE/DEAD cell Staining Kit (Enzo Life Sciences AG, Lausen, Switzerland). Then, 1 × 10⁴ cells were seeded on either BHM or MCM (the porous layer faces up) membrane in 200 μl of DMEM. After 1, 3, and 7 days of culture, the cells were stained with 100 μl of staining solution at 37°C for 15 min and observed under a fluorescence microscope immediately after the staining.

To assess the viability of GC, PDLC, and L-929 rods, samples were stained with the Live/Dead Cell Staining Kit (Enzo Life Sciences AG, Lausen, TX, United States) following the manufacturer’s guidelines in a 24 well plate after the 24 h incubation period (Janjić et al., 2018a (link)). The vitality of the rods was analyzed using fluorescence microscopy for green and red, with a B-2A filter (excitation filter wavelengths: 450–490 nm), respectively. Vital cells became visible as green while the dead cells were red.