Live dead cell viability assay kit

To investigate whether UA could potentiate the efficacy of gemcitabine for causing apoptosis in pancreatic cancer cells, we used a LIVE/DEAD cell viability assay kit (Invitrogen), which is used to determine intracellular esterase activity and plasma membrane integrity and was described previously. AsPC-1, MIA PaCa-2, and Panc-28 cells (5,000 per well) were incubated in chamber slides, pretreated with 20 μM of UA for 8 hours. Cell were washed with PBS to remove UA and then treated with 100 nM of gemcitabine for 24 hours. The cells were then stained with the assay reagents for 30 minutes at room temperature, and cell viability was determined by counting live (green) and dead (red) cells under a fluorescence microscope.

To evaluate the cell viabilities of the skeletal muscle tissue in the biohybrid robot, we stained it with 0.1% calcein AM and 0.2% ethidium homodimer (Live/Dead cell viability assay kit L3224; Invitrogen). To visualize α-actinin and the cell nuclei of the skeletal muscle tissue, the skeletal muscle tissue formed on the flexible substrate was washed using phosphate-buffered saline without Mg²⁺ and Ca²⁺ [PBS(−), Cell Science & Technology Institute, Inc.], fixed with 4% paraformaldehyde (Muto Pure Chemicals Co., Ltd.), permeabilized using 0.1% Triton X-100 (Alfa Aesar) in PBS(−) for 20 min, and then blocked with 2.5% bovine serum albumin (BSA, Sigma-Aldrich Co., LLC.) in PBS(−) overnight. Afterwards, we incubated the skeletal muscle tissue on the flexible substrate with PBS(−) containing 2.5% BSA and 0.1% monoclonal anti-α-actinin antibody (Sigma-Aldrich) at 4 °C, overnight. The skeletal muscle tissue was rinsed with PBS(−) and incubated using 0.1% Alexa Fluor conjugated secondary antibody (Thermo Fisher Scientific, Inc.) for 2 h at room temperature. After rinsing it with PBS(−) again, we stained the cell nuclei with 0.1% Hoechst 33342 (Invitrogen).

After 72 h of culture, the cells were fixed in 4% paraformaldehyde for 20 min, permeabilized in 0.5% Triton X-100 in PBS for 30 min, and then blocked in 5% bovine serum albumin (BSA) in PBST (0.1% Tween 20) for 30 min. Then the cells were incubated with 5 μg/ml fluorescein isothiocyanate (FITC)-phalloidin for 30 min at room temperature, whereas the nuclei were stained with DAPI. Phase-contrast and fluorescence images were captured using an inverted fluorescence microscope (Olympus IX73, Tokyo, Japan) and a confocal laser-scanning microscope (Eclipse Ti2, Nikon).
Live/dead assay was performed using a Live/dead cell Viability Assay Kit (Invitrogen). Nonfluorescent calcein-AM is hydrolysed by the live cells into green fluorescent calcein, whereas ethidium homodimer-1 can only pass through the membrane of the dead cells. The cells were rinsed twice with PBS before the fluorochromes were added and incubated for 45 min. Fluorescence images were captured using an inverted fluorescence microscope (Olympus IX73, Tokyo, Japan).

Human BM-MSCs were seeded at a density of 5,000 cells/cm² in 12-well plates and cultured in the growth medium or the growth medium supplemented with strontium at doses of 0.1 mM, 1 mM, and 10 mM, respectively. On days 1, 3, and 7, a Live/Dead® Cell Viability Assay Kit (Invitrogen, USA) was used to monitor cell viability. The samples (n = 3 for each group) were gently washed with PBS for two times and then incubated in the working solution for 30 mins at 37°C. Cell viability was observed by an inverted fluorescence microscope (Olympus Corporation, Japan).