Calam

PPIs (100 IEQ) were washed with phosphate buffered saline and incubated for 30 min in Calcein AM (CalAM, Invitrogen, Carlsbad, Califonria, USA, cat#C1430) and propidium iodide (PI, Invitrogen, cat#P3566, Carlsbad, Califonria, USA) for staining of living and dead cells, respectively^{23 (link)}. After washing, the viability of CalAM/PI-stained islets was evaluated using a microplate reader (Tecan Infinite F200; Tecan, Chapel Hill, North Carolina, USA) and calculated using the following equation: CalAM-positive cells/(CalAM-positive cells + PI-positive cells) × 100.

A LIVE/DEAD viability/cytotoxicity kit (L3224; Thermo Fisher Scientific) was used to assess cell viability in chondrocyte‐laded alginate beads according to manufacturer's instructions. Briefly, chondrocyte‐laden alginate beads were washed in PBS, followed by incubation in 0.5 μL/mL Cal‐AM and 2 μL/mL Eth‐D (Thermo Fisher) in PBS at 37°C for 1.5 h. Beads were washed again in PBS and imaged using light sheet or confocal microscopy. Live and dead cell quantification was performed in ImageJ v1.47 for Mac using the “process > find maxima” function.
Cell quantification in alginate beads cultured in proliferation and chondrogenic medium was performed using DAPI staining. Unfixed chondrocyte‐laden alginate beads were covered in Tissue‐Tek, snap frozen, and cut in 20 μm sections using a cryostat. Following the application of mounting medium (Fluoroshield Mounting Medium with DAPI; Abcam), beads (n = 3 per group) were imaged on a Nikon Eclipse 80i confocal microscope. Cells were counted in NIS‐Elements AR software (v 3.2; Nikon Instruments Europe B.V). Both the total number of cells and percent (%) cells per bead were calculated per section.

Triclosan was obtained from Shanghai Baidi Biody-Bio Co., Ltd. Hoechst, Cal-AM, Eth-1, Fluo-3/AM, mito-Tracker, mito-SOX, tetramethylrhodamine methyl ester (TMRM) and DAPI were purchased from Thermo Fisher Scientific, Inc. Dihydroethidium dye was purchased from the Beyotime Institute of Biotechnology. Tempol, 3-MA and acetylcysteine (NAC) were purchased from Sigma Aldrich; Merck KGaA. Tempol (0.5 and 1 mM) is a radical scavenger that was used to test the effect of ROS levels on cytotoxicity induced by Triclosan (20 µM) in the lactate dehydrogenase (LDH) release assay. S3I-201 was purchased from EMD Millipore. S3I-201 (10 and 20 µM) is a STAT3 inhibitor that was used to detect the effect of STAT3 activity change on cytotoxicity induced by Triclosan (20 µM) in the LDH release assay. Anti-p-STAT3 (Y705, #9131, 1:1,000), anti-STAT3 (#4904, 1:1,000), anti-p-AMPK (Thr172, #2535, 1:1,000), anti-AMPK (#2532, 1:1,000) and anti-p62 (#88588, 1:1,000) were purchased from Cell Signaling Technology, Inc., Bcl-2 (ab196495, 1:1,000) antibody was purchased from Abcam and LC3 (L7543, 1:1,000) antibody was purchased from Sigma Aldrich; Merck KGaA.