Pluronic f 127

Rhod-2 acetoxymethyl ester (Rhod-2, AM; GeneCopoeia, United States) was used to measure the mitochondrial Ca²⁺ concentrations. According to our previous experience, the fluorescence value of Rhod-2 AM approximately represented the fluorescence value of mitochondrial Ca^2+[11,24]. The day before the experiment, 1 x 10⁵ cells were plated in 10-mm confocal dishes (Nest Biotechnology, China). After overnight incubation, the growth medium was removed from the cell plates, and added with 500 μL medium with 4 μM Rhod-2 AM and 0.1% Pluronic^® F-127 (GeneCopoeia) and allowed to incubate at 37 °C in humidified air (containing 5% CO₂) for 2 h. Next, D-Hanks balanced salt solution (Hangzhou Genom Biochemical and Pharmaceutical, China) was used to wash the cells three times, followed by addition of 500 μL Hoechst-33342 and 200 nmol/L MitoTracker Green (Beijing Beyotime Institute of Biotechnology), and allowed to incubate in the same environment for 30 min of cultivation. Confocal microscopy confirmed that the vast majority of Rhod-2 AM fluorescence was associated with the mitochondria. Furthermore, cells were incubated in 96-well plates with Rhod-2 AM and F-127 in the same process and observed by VarioskanFlash (Thermo Fisher Scientific) with the illumination at excitation 552 nm and emission 581 nm to confirm the results obtained from confocal microscopy.