Mitochondrial membrane potential assay kit with jc 1

RPMI Medium 1640 basic(1X), fetal bovine serum (FBS), horse serum (HS), neurobasal™ medium, B-27™ supplement (50 × ), glucose solution, L-glutamine (200 mM) were purchased from Gibco^® (Thermo Fisher Scientific, NY, United States). Recombinant Human β-Nerve Growth Factor (NGF, 98% pure) was obtained from Peprotech (Cranbury, NJ, United States). Amyloid β-protein fragment 25–35 (Aβ_25–35, 97% pure) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), Triton X-100, paraformaldehyde, goat serum and 4-6-diamidino-2-phenylindole (DAPI) were from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). A lactate dehydrogenase (LDH) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The reactive oxygen species (ROS) assay kit and mitochondrial membrane potential (MMP) assay kit with JC-1 were obtained from the Beyotime Institute of Biotechnology (Nanjing, China). The anti-MAP2 (phospho S136) antibody, goat anti-rabbit IgG H&L (Alexa Fluor^® 488) were obtained from Abcam (Cambridge, United Kingdom).

The mitochondrial membrane potential was detected with mitochondrial membrane potential assay kit with JC-1 (C2006, Beyotime, China). Treated OS cells with atezolizumab for 24 h, and replaced the medium with fresh medium. Then incubated with JC-1 working solution in a CO₂ incubator for 20 min. After incubation, removed the staining solution and washed with JC-1 staining buffer twice. OS cells were imaged using confocal microscopy and the change in mitochondrial membrane potential (Δψm) was reflected by the ratio of green fluorescence to red fluorescence (ratio of JC-1 monomers/JC-1 aggregates).

Thiazolyl Blue Tetrazolium Bromide (MTT) and 2′,7′-Dichloroflurescin diacetate (DCFH-DA) were obtained from Sigma (USA). Penicillin, streptomycin and 0.05% trypsin were purchased from Invitrogen (USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Hyclone (USA). Fetal bovine serum (FBS) was from Corning (USA) and Tiron was from SigmaeAldrich (USA). A transferasemediated uridine nick end labeling (TUNEL) detection kit was obtained from Roche (Germany). Mitochondrial membrane potential assay kit with JC-1 and Hoechst33342 staining assay were from Beyotime Technology (China). Earle’s balanced salt solution was from Aladdin (China). The primary antibodies against p-Akt, and Cleaved-caspase-3 were from Cell Signaling Technology (USA) and antibodies against P53, Bcl-2 and Bax were from Abcam (USA). Antibodies against β-actin, GAPDH and secondary antibodies were purchased from Beijing Zhong Shan Jinqiao Biological Technology Co., Ltd (China). LY294002 was obtained from Selleck (USA). Freeze-dried powder of Qishenkeli (QSKL) was prepared by College of TCM, Beijing University of Chinese Medicine. QSKL and LY294002 were dissolved in DMSO or DMEM for in vitro assay as storage concentration.

The mitochondrial membrane potential of the oocytes was evaluated using a mitochondrial membrane potential assay kit with JC-1 (Beyotime Institute of Biotechnology, China). Oocytes were exposed to 10μM JC-1 in 100μl working solution at 38.5°C in 5% CO₂ for 20 min, after which they were washed with washing buffer to remove surface fluorescence, and then mounted on glass slides using D-PBS for microscopy. Laser excitation was set at 488 nm for green and 525 nm for red fluorescence, respectively. The fluorescence intensity in each oocyte was measured under a fluorescence microscope (Olympus, Tokyo, Japan) with the same scan settings for each sample. The fluorescent images were saved as graphic files in TIFF format. The normal fluorescence pixel intensities of each oocyte were analyzed using ImageJ software (version 1.50; National Institutes of Health, Bethesda, MD, USA). The ratio of green to red fluorescence pixels was used to analyze mitochondrial membrane potential.

The production of ROS was measured by an ROS assay kit (Cat No. S0033S, Beyotime, China). A549 cells were incubated with 10 μM DCFH-DA for 20 min at 37°C in the dark. Then, the cells were gently washed 3 times with warm buffer. The cells were observed under a fluorescence microscope, and the fluorescence intensity was measured at the excitation/emission maxima at 488/525 nm using a fluorescence microplate reader.
MMPs were assessed using the Mitochondrial Membrane Potential Assay Kit with JC-1 (Cat No. C2006, Beyotime, China). After experimentation, the cells were treated with JC-1 staining working solution in a dark environment for 20 min and washed twice with cold staining buffer. A fluorescence microplate reader was used to detect JC-1 monomers at 490/530 nm and JC-1 aggregates at 525/590 nm.

HL-7702 cells were provided by Shanghai Huiying Biological Technology Co., Ltd (Shanghai, China). T-2 toxin was purchased from Beijing Puhuaren Technology Development Co., Ltd (Beijing, China). Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), collagenase type IV, and penicillin–streptomycin was purchased from Gibco (Thermo Fisher, Shanghai, China). Cell Counting Kit-8 (CCK-8), Annexin V-FITC/PI apoptosis detection kit, ATP assay kit, ROS assay kit, and mitochondrial membrane potential assay kit with JC-1 were purchased from Beyotime Biotechnology (Beyotime, Haimen, China). All real-time PCR reagents were purchased from TaKaRa (Dalian, China). All other chemicals, if not stated, were purchased from Sigma.

Mitochondrial membrane potential assay kit with JC-1 (Beyotime, China) was purchased to evaluate the mitochondrial membrane potential, the fluorescence intensity of the JC-1 monomers/aggregates (green fluorescence for monomer, red fluorescence for aggregate) were taken by fluorescence microscopy (Leica), following the instructions of JC-1 assay kit. Digital images were analyzed with ImageJ software (NIH, Bethesda, MD, USA). The calculation results of the red/green fluorescence ratio were for assessing mitochondrial membrane potential.