Mitochondrial permeability transition pore assay kit

mPTP (mitochondrial permeability transition pores) was detected using a mitochondrial permeability transition pore assay kit (Beyotime). Briefly, brown and beige adipocytes were washed twice with PBS and loaded with calcein-AM for 15 min at 37 ℃, following by additional incubation with 1 mM CoCl₂ for 1 h. After washing with PBS, cells were imaged on a confocal microscope with an argon laser at 488 nm and an emission filter at 525 nm (Leica).

Blood, tissue, or cell samples were collected and used to measure IL-1α, INF-γ, TNF-α, IL-6, and IL-1β levels using IL-1α, INF-γ, TNF-α, IL-6, and IL-1β ELISA kits (Nanjing Jiancheng Biological Engineering Institute, Nanjing, China) following the manufacturer's instructions. ROS production was evaluated by measuring ROS levels kits (S0033S, Beyotime Biotechnology) following the manufacturer's instructions. Calcein AM/CoCl2 assay and JC-1 disaggregation were evaluated by mitochondrial permeability transition pore assay kit (C2009S, Beyotime Biotechnology) and enhanced mitochondrial membrane potential assay kit with JC-1 (C2003S, Beyotime Biotechnology).

The opening of mPTP was detected using the Mitochondrial Permeability Transition Pore Assay kit (Beyotime, China). In brief, 3 h after the indicated treatments, cell-permeable calcein acetoxymethyl ester and CoCl₂, the quencher of calcein fluorescence, were applied at 37 °C for 30 min to selectively label mitochondria. The green fluorescent signal of calcein was then visualized using a fluorescence microscope (DMI6000, Leica, Germany) and quantified by ImageJ.

Doxorubicin hydrochloride (Dox·HCl) was purchased from Dalian Meilun Biotech Co., Ltd. (Dalian, China). GA was purchased from J&K Co. Ltd. (Shanghai, China). Initiator ABIK‐PDS was synthesized according to a previous report. POSS‐NH2 was purchased from Hybrid Plastics (Hattiesburg, USA). Matrigel was purchased from BD Biosciences (Bedford, USA). 3‐(4,5‐dimethyl‐2‐tetrazolyl)‐2,5‐diphenyl‐2H tetrazolium bromide (MTT), 4’,6‐diamidino‐2‐phenylindole (DAPI) and reduced GSH, were purchased from Sigma‐Aldrich (St. Louis, MO, USA). MitoTracker Green was purchased from Invitrogen (Carlsbad, CA, USA). BCA Protein Assay Kit, SDS‐PAGE Gel Quick Preparation Kit, Mitochondrial Permeability Transition Pore Assay Kit, Cell Mitochondria Isolation Kit and Reactive Oxygen Species Assay Kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). FITC Annexin V Apoptosis Detection Kit with 7‐AAD were purchased from BioLegend. Comonomer HPMA and N‐methacryloylglycylglycyl‐hydrazide‐doxorubicin (MA‐GG‐NHN = Dox) were synthesized according to previous method.^[21] All other chemicals and reagents were of analytical grade. All other chemicals were bought from Energy Chemical (Shanghai, China).

Mitochondrial quality was assessed using the Mito-Tracker Red CMXRos Assay Kit (M7512, Invitrogen). MMP was detected using the MitoProbe™ JC-1 Assay Kit (M34152, Invitrogen). mPTP opening was detected using the Mitochondrial Permeability Transition Pore Assay Kit (C2009S, Beyotime). All operations were performed following the manufacturers’ instructions.

For the detection of mitochondrial membrane potential (∆Ψm, MMP), NRCMs were washed with PBS and then stained with JC-1 working solution (Beyotime) for 20 min at 37 °C. Five images of each sample were photographed using fluorescence microscopy (Leica, DM2500) and analysed by ImageJ software (1.48v). The relative ratio of JC-1 aggregate (red)-to-monomer (green) fluorescence intensity was used to evaluate the proportion of depolarized mitochondria [30 (link)].
For the examination of mitochondrial permeability transition pore (mPTP), NRCMs were stained with a Mitochondrial Permeability Transition Pore Assay Kit based on the description of manufacturer (Beyotime). The relative calcein green fluorescence intensity in mitochondria was used to judge the degree of mPTP opening. All values of fluorescence intensity were normalized to controls. Five visual fields in each sample were randomly chosen to quantify the JC-1 or mPTP fluorescence intensity.