Mmp detection kit

AST was dissolved in dimethyl sulfoxide (Sigma-Aldrich, St Louis, MO, USA) to yield a 10 mM stock solution as our previously described 11 (link),12 . Rat lung epithelial-T-antigen negative (RLE-6TN) cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cell line was derived from AECs-II isolated from a 56 day old male F344 rat using airway perfusion with a pronase solution, which exhibits characteristics of AECs-II such as lipid-containing inclusion bodies and expression of cytokeratin 8 and 19. Pharmaceutical grade BLM was purchased from Nippon Kayaku Co., Ltd. (Tokyo, Japan). MMP detection kit was purchased from Beyotime Institute of Biotechnology (Haimen, China). Antibodies against Bcl-2, Bcl-XL, Bax, Bad, cytochrome c (Cyt c), Puma, Nuclear factor erythroid 2-related factor 2 (Nrf-2), P53 were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA).

The mitochondria were extracted from the hippocampal tissues of mice (n = 4 per group) using a mitochondrial extraction kit (Solarbio, Beijing, China). The homogenization of hippocampal tissue was performed in an ice-cold bath at 0 °C, followed by centrifugation at 4 °C to extract the mitochondria, which were then rapidly transferred and stored at -70 °C. The proteins were measured by the bicinchoninic acid (BCA) method and then diluted to the same concentration. JC-1 staining working solution was prepared following the instructions provided in the MMP detection kit (Beyotime Biotechnology, Shanghai, China) and was subsequently mixed with the extracted hippocampal mitochondria. The preparation and addition of JC-1 working solution were also performed in a sterile ice-cold bath at 0 °C, followed by centrifugation at 4 °C. The fluorescence intensities of the JC-1 polymer and monomer were measured at 525/590 nm and 490/530 nm, respectively.

The MMP was determined with an MMP detection kit (C2006, Beyotime, Shanghai, China). The chemiluminescence signal was detected using a fluorescence microplate reader (BioTek Instruments). CCCP (Carbonyl cyanide m-chlorophenylhydrazone) (a membrane uncoupling chemical) was used as a positive control. The results were presented as relative fluorescence intensity and were normalized to that of the control group.

Cells cultured in 24-well plates were infected and/or transfected as indicated. After different treatment, the MMP of treated cells was measured by an MMP Detection Kit (C2006, Beyotime) according to the manufacturer’s instructions. For fluorescence microscope observation, cells were rinsed with pre-warmed PBS for one time and incubated with JC-1 staining solution (5 μg/mL) for 20 min at 37°C. Cells were then rinsed twice with cold JC-1 staining buffer, and fluorescence intensity of both JC-1 monomers (Ex/Em = 514/529 nm) and JC-1 aggregates (Ex/Em = 585/590 nm) were detected using an inverted fluorescent microscope (AXIOVERT A1, Zeiss, Oberkochen, Germany). For flow cytometry analysis, treated cells were harvested and rinsed with PBS twice. After corresponding treatment, the fluorescence intensity of JC-1 monomers and JC-1 aggregates were analyzed using CyAn ADP7 flow cytometer.

MC3T3-E1 cells were inoculated in 6-well plates at a density of 1.0 × 10⁵ cells per well. After incubation to 60% confluence, they were incubated with Dex with or without ADSCs-Exos. The MMP detection kit (Beyotime, China) was used to measure MMP levels in different groups of cells following the manufacturer's instructions. Briefly, the cells were coincubated with the JC-1 staining working solution at 37°C for 30 min and rinsed with a precooled JC-1 staining buffer. The cells were visualized by fluorescence microscopy.

Mitochondrial membrane potential was detected using a MMP detection Kit (C2006, Beyotime, China). Briefly, after different treatments, cells were harvested and 1 mL JC-1 solution was added and incubated at 37°C for 20 min. After the incubation, cells were washed three times with PBS, resuspended, and analyzed by flow cytometry.

Mitochondrial membrane potential (MMP) was determined using the JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide) MMP detection kit (Beyotime). JC-1 is a cationic fluorescent dye probe (green as monomer/red as aggregates) which accumulates in mitochondria in a potential-dependent manner. Cells with functional mitochondria incorporate JC-1 leading to the formation of JC-1 aggregates, which show a red spectral shift resulting in higher levels of red fluorescence emission measured in the red (fluorescence, FL-2 channel) and green monomers (detectable in FL-1 channel). Cells with collapsed mitochondria contain mainly green JC-1 monomers. These were performed as previous described [23 (link)]. All of the standards, controls and samples were read in duplicate.