Journal: Antioxidants

Article Title: Development of an AAPH-Induced Oxidative Stress Model in Bovine Mammary Epithelial Cells and Investigation of Its Molecular Mechanisms

doi: 10.3390/antiox15040460

Figure Lengend Snippet: ( A ) Representative fluorescence images of intracellular ROS levels detected using the DCFH-DA probe. Green fluorescence intensity reflects the relative level of reactive oxygen species. Scale bar = 50 μm. ( B ) Quantitative analysis of relative ROS fluorescence intensity. Data are presented as mean ± SD from three independent experiments. NC: negative control (untreated cells); PC: positive control (cells treated with the ROS-positive control reagent provided in the kit); AAPH: cells treated with 1000 μmol/L AAPH for 6 h; NAC: cells pretreated with N-acetylcysteine prior to AAPH treatment. ( C ) Quantitative analysis of the relative ratio of red (J-aggregates) to green (JC-1 monomer) fluorescence intensity measured by the JC-1 assay. A decrease in this ratio indicates loss of mitochondrial membrane potential. (ns: p > 0.05; ** p < 0.01; and *** p < 0.001). ( D ) Representative fluorescence images of JC-1 staining. Red fluorescence indicates JC-1 aggregates in mitochondria with high membrane potential (healthy mitochondria), while green fluorescence indicates JC-1 monomers in mitochondria with low membrane potential (mitochondrial injury). The merged images show colocalization. Scale bar = 50 μm.

Article Snippet: AAPH, N-Acetylcysteine (NAC), the Cell Counting Kit-8, ROS Assay Kit, and JC-1 Mitochondrial Membrane Potential Assay Kit were purchased from MedChemExpress (MCE) (Monmouth Junction, NJ, USA).

Techniques: Fluorescence, Negative Control, Positive Control, Membrane, Staining

Journal: Ecotoxicology and environmental safety

Article Title: Mechanistic insights into severe pulmonary inflammation caused by silica stimulation: The role of macrophage pyroptosis.

doi: 10.1016/j.ecoenv.2023.114975

Figure Lengend Snippet: Fig. 2. Silica exposure induces mitochondrial depolarization and disrupts energy homeostasis in RAW-ASC cells. RAW-ASC cells were primed with LPS for 6 h then treated with nano-silica, micro-silica, and ATP for 4 h, respectively. (A) Representative images of JC-1 staining showing the alteration of mitochondrial membrane potential between groups. Red, JC-1 aggregates; Green, JC-1 monomers. Scale bar = 50 µm. (B) Quantitative analysis and comparison of fluorescence intensity in A between groups (n = 6). (C) Measurement and comparison of intracellular ROS content between groups (n = 6). (D) Measurement and comparison of intracellular ATP content between groups (n = 4). ns, not significant, *P < 0.05, **P < 0.01.

Article Snippet: The mouse macrophage cell line RAW264.7 cells stably expressing ASC (RAW-ASC) were obtained from Invivogen (San Diego, CA, USA); Dulbecco’s modified Eagle’s medium of high glucose (DMEM), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody, and goat antirabbit IgG-HRP conjugated secondary antibody were obtained from Servicebio (Wuhan, China); Silica nanoparticles, penicillinstreptomycin, mitochondrial Membrane Potential Assay Kit with JC-1, and adenosine triphosphate (ATP) were purchased from Solarbio (Beijing, China); Brilliant blue G (BBG), lipopolysaccharides (LPS), reactive oxygen assay kit, calcium content chromogenic assay kit, ATP assay kit, and Calcein/PI cell assay kit were purchased from Beyotime (Shanghai, China); KCl was obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China); Fetal bovine serum (FBS) was obtained from BI (Israel); Silica particles of micro-size, and fast green FCF were obtained from Sigma-Aldrich (St. Louis, MO, USA); Primary antibodies of NLRP3, P2X7, Pannexin-1, and IL-1β were purchased from ABclonal (Wuhan, China).

Techniques: Staining, Membrane, Comparison, Fluorescence