Live dead assay kit

The GSK1016790A (Millipore Sigma, Burlington, MA, USA, G0798) and RN-1734 (Millipore Sigma, R0658) were used as an agonist and an antagonist for TRPV4 ion channels, respectively. These modulators were chosen based on their level of selectivity [29 (link),30 (link)]. For activating TRPV4 channels, GSK1016790A was added to the macrophage media with a concentration of 500 nM. The concentration was chosen based on published literature data based on cell viability and efficiency [31 (link),32 (link)]. For blocking TRPV4 channels, RN-1734 was added into the media with 10 µM concentration following concentration optimization analysis. Briefly, to identify the optimal concentration for the TRPV4 antagonist, the macrophages were cultured with 10 nM, 1 mM, and 10 µM RN-1734, and cell viability was measured using a Live-Dead Assay kit (Life Technologies, Carlsbad, CA, USA). Following incubating macrophages for 48 h with various antagonist concentrations, the cells were washed with dPBS and cultured with 2 mM of Calcein AM and 4 mM ethidium homodimer-1 dyes for 2.5 h at 37 °C and then fixed with 4% paraformaldehyde (Sigma, USA) for 30 min. The live and dead cells were then visualized using fluorescence microscopy at 490/525 nm and 557/576 nm excitation/emission wavelengths to visualize live (green) and dead (red) cells, respectively.

Cell viability assay was performed on the scaffolds and the coverslips after 48 h using the Live/Dead assay kit (Life Technologies). The optimal dye concentrations of Calcein AM, the live cell label and EthD-T, the dead cell label was optimized to 2 µM each. After 48 h the scaffolds were sliced and the sections and the coverslips were then used in image analysis under a fluorescence microscope (Inverted Laboratory Microscope, Leica DM IL LED) and the live cell and dead cell numbers were determined.

Cell viability was determined using a Live/Dead assay kit (Life Technologies, Scoresby, VIC, Australia) as per manufacturer's instructions. Briefly, hBMSCs were encapsulated in hydrogels and transferred at the appropriate timepoint to DPBS for 15 ​min to limit undesirable serum esterase activity. Staining solution containing 2 ​μM calcein AM (0.05% v/v) and 4 ​μM EtD-1 (0.2% v/v) was prepared in sterile DPBS. Hydrogel constructs were submerged in the staining solution and incubated for 30 ​min at room temperature in the dark. Cells were imaged at three predetermined areas per sample using a Nikon Eclipse Ts2 Inverted Routine Microscope equipped with a Lumenera Infinity3-3UR camera using Infinity analysis software. Each condition was imaged in triplicate and analysed using ImageJ software (NIH, USA) by manual counting of live and dead cells.