Mptp detection kit

A mitochondrial permeability transition pore (mPTP) detection kit (Beyotime, Shanghai, China) was used to detect the opening degree of mPTP in each group of cells, reflecting the concentration of calcium ions in mitochondria. Cells in each group were seeded in a confocal hole plate (NEST, Wuxi, Jiangsu, China), washed twice with PBS, for 1 min each time. 1 ml Calcein AM staining solution was added to each hole, and incubated in a 5% CO₂ incubator at 37 °C for 30 min in the dark. After staining and incubation, the preheated culture medium at 37 °C was replaced, and then incubated at 37 °C for 30 min again. The laser confocal microscope was excited at a 488 nm wavelength. Other related treatment reagents were used to set blank and control groups: quenching working solution (Calcein AM containing 10% CoCl₂), calcium overload solution (Calcein AM containing 10% CoCl₂ and 5% lonomycin), and ARL2-siRNA treatment.

We determined MMP using the mitochondrial membrane potential detection kit (JC1, Beyotime Biotech, China) following specific protocols. Later, we tested the opening degree of the MPTP using the MPTP Detection Kit (Beyotime Biotech, China), following specific instructions (http://www.beyotime.com/index.htm). Flow cytometry was used to analyze the MMP and MPTP opened of the macrophages derived from peripheral blood [27 (link)–29 (link)].