Lysotracker green

RAP and PLGA (MW 90000, 50:50) were purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, Chia). 1,19-Dioctadecyl-3,3,39,39-tetramethylindodicarbocyanine perchlorate (DiD) was purchased from Biotium Inc. (Fremont, US). DiO, DAPI, Cell Total Protein Extraction kits and Membrane Protein Extraction kits were supplied by Beyotime Institute of Biotechnology (Jiangsu, China). The CellTiter 96^TM AQueous One Solution Cell Proliferation Assay (MTS) was purchased from Thermo Fisher Scientific (SanJose, CA, USA). Lipopolysaccharide (LPS) was purchased from Solarbio (Beijing, China). The mouse glycoprotein ELISA kit, mouse TNF-α ELISA kit and mouse IL-6 ELISA kit were purchased from Wuhan Colorful Gene Biological Technology Co., Ltd. (Wuhan, China). LysoTracker Green was purchased from Yeasen Biotech Co. Ltd. (Shanghai China). Ultrapure water with a resistivity of 18.2 MΩ·cm was used throughout the experiments.

LysoTracker Green (Yeasen Biotech Co., Ltd., Shanghai, China) and MitoTracker Deep Red (Thermo Fisher Scientific, Waltham, MA, United States) double staining was used for the evaluation of mitophagy. Briefly, SCCs were incubated in α-MEM supplemented with 50 nM LysoTracker Green and 100 nM MitoTracker Deep Red at 37°C for 40 min. After two washes, the medium was replaced with fresh α-MEM, and images of live cells were captured using a confocal microscope (Olympus, Tokyo, Japan). Colocalization was quantified using ImageJ.

After different treatments, cells were washed with fresh DMEM and incubated with 75 nM LysoTracker Green for 1 h at 37 °C (Yeasen Biotech, Shanghai, China). Nuclei were stained with Hoechst 33,258 (Servicebio, Wuhan, China), and images were captured with a fluorescence microscope.

ER‐tracker and Mito‐Tracker double staining were used to evaluate the morphology of mitochondria and ER‐mitochondria colocalization in MSCs as previously reported.^[10] Briefly, MSCs were incubated in α‐MEM supplemented with 1 × 10⁻³m ER‐Tracker green (Thermo Fisher Scientific, MA, USA) and 100 × 10⁻⁹m Mito‐Tracker deep red (Thermo Fisher Scientific) at 37 °C for 30 min.
For the mitophagy assay, Lyso‐Tracker green (Yeasen Biotech Co., Ltd., Shanghai, China) and Mito‐Tracker deep red double staining were used to evaluate mitophagy. MSCs were incubated in α‐MEM supplemented with 50 × 10⁻⁹m Lyso‐Tracker green and 100 × 10⁻⁹m Mito‐Tracker deep red at 37 °C for 40 min. After washing, the medium was replaced with fresh α‐MEM. For the autophagy flux assay, MSCs were transfected with adenoviruses expressing mRFP‐GFP‐LC3 (GENECHEM, Shanghai, China) for 72 h. The nuclei were stained with 1 µg mL⁻¹ Hoechst 33342 (Sigma‐Aldrich).
Live‐cell images were captured using a Nikon laser scanning confocal microscope (A1 Plus, Nikon, Japan). Thresholded images were evaluated using the “Analyze Particles” function in ImageJ (Media Cybernetics, USA) to obtain the number, perimeter, and area of mitochondrial fragments per cell. Quantification of colocalizations was performed with the Colocalization Plugin in ImageJ 1.52v.