Tmrm detection kit

Human kidney proximal tubular epithelial cells (HK-2) were a cell line purchased from the American Type Culture Collection (ATCC, USA). Antibodies were from the following sources: polyclonal anti-TLR4 from Abcam (USA). Monoclonal anti-PGC-1α, monoclonal anti-caspase-3, and NF-κB p65 were from Cell Signaling Technology (Boston, USA). Polyclonal phospho-NF-κB p65 (ser536) antibody was from Bioworld (USA), and polyclonal anti-cytochrome C was from Proteintech (Wuhan, China). Beta-actin and all secondary antibodies for Western blot and immunofluorescence were from Proteintech (Wuhan, China). TAK242 was from MedChem Express (USA), and parthenolide was from Sigma (USA). Plasmids containing pcDNA4 myc PGC-1α (pcDNA4/PGC-1α) was bought from Addgene (USA). Lipofectamine 2000, MitoTracker Red CMXRos, MitoSOX, and TRIzol were purchased from Invitrogen (USA). PrimeScript™ RT reagent Kit with gDNA Eraser and SYBR® Premix Ex Taq™ (Tli RNase H Plus) were from TaKaRa (Japan). Annexin V-FITC Apoptosis Detection Kit was from Beyotime (Shanghai, China). TMRM Detection Kit was from Genmed Scientifics Inc. (USA). Other reagents, including DMEM/F12 medium, bovine serum albumin (FBS), and trypsin, were obtained from Gibco (USA).

Mitochondrial △Ψ_m of cells was assessed by TMRM Detection Kit (Genmed Scientifics Inc., USA). HK-2 cells plated on 24-well culture dishes were harvested after trypsinization when they were seeded at 70% confluence, and then the cells were stained with TMRM dyeing liquid according to the manufacturer's instruction. For 20 min incubation in the dark at 37°C, mitochondrial △Ψ_m of cells was examined by a FACSCalibur flow cytometer (BD Biosciences, San Jose, USA). The fluorescence intensity of TMRM was monitored at 575 nm.