emission fluorescence signals  (Oxford Instruments)

Journal: Antioxidants

Article Title: MOMAST ® Downregulates AQP3 Expression and Function in Human Colon Cells

doi: 10.3390/antiox14010026

Figure Lengend Snippet: ROS content in HCT8 cells exposed to MOMAST ® . ROS content was measured using dihydrorhodamine-123 fluorescence in cells left under basal conditions or treated with MOMAST ® as described in Methods. As a positive control, cells were treated with the oxidant tert -Butyl hydroperoxide (tBHP). Untreated cells (CTR) are a blank control. Data are shown as means ± Standard Error Means (S.E.M.) of six independent experiments and analyzed with one-way ANOVA followed by Dunnett’s multiple comparisons test ( **** p < 0.0001 vs. CTR; ## p < 0.01 vs. tBHP; #### p < 0.0001 vs. tBHP).

Article Snippet: Then, the fluorescence emission signal was measured using the FLUOstar Omega Microplate Reader (BMG Labtech, Ortenberg, Germany).

Techniques: Fluorescence, Positive Control, Control

Journal: Antioxidants

Article Title: MOMAST ® Downregulates AQP3 Expression and Function in Human Colon Cells

doi: 10.3390/antiox14010026

Figure Lengend Snippet: ( a ) Real-time PCR analysis of AQP3 mRNA expression levels. MOMAST ® treatment did not alter the AQP3 transcript levels in HCT8 cells. Data are shown as means ± Standard Error Means (S.E.M.) of three independent experiments and analyzed with one-way ANOVA followed by Dunnett’s multiple comparisons test. ( b ) Confocal immunofluorescence visualization of AQP3 in HCT8 cells. Cells were left under basal conditions or exposed to increasing concentrations (250, 500, and 1000 µg/mL) of MOMAST ® as described in Methods. Confocal images show a slight but relevant reduction in AQP3 fluorescence signal.

Article Snippet: Then, the fluorescence emission signal was measured using the FLUOstar Omega Microplate Reader (BMG Labtech, Ortenberg, Germany).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Fluorescence

Journal: Antioxidants

Article Title: MOMAST ® Downregulates AQP3 Expression and Function in Human Colon Cells

doi: 10.3390/antiox14010026

Figure Lengend Snippet: Evaluation of the HyPer-3 sensitivity to H 2 O 2 exposure by analyzing the HyPer-3 fluorescence ratio. ( a ) A representative trace showing the HyPer-3 fluorescence ratio in transfected HCT8 cells stimulated with external H 2 O 2 at increasing concentrations (25, 50, and 100 μM). ( b ) A significant increase in normalized HyPer-3 fluorescence ratio intensity was recorded at 50 and 100 μM H 2 O 2 external stimuli. Data are shown as means ± Standard Error Means (S.E.M.) of six independent experiments and analyzed with one-way ANOVA followed by Dunnett’s multiple comparisons test (** p < 0.01 vs. basal; **** p < 0.0001 vs. basal).

Article Snippet: Then, the fluorescence emission signal was measured using the FLUOstar Omega Microplate Reader (BMG Labtech, Ortenberg, Germany).

Techniques: Fluorescence, Transfection

Journal: Antioxidants

Article Title: MOMAST ® Downregulates AQP3 Expression and Function in Human Colon Cells

doi: 10.3390/antiox14010026

Figure Lengend Snippet: Evaluation of the HyPer-3 sensitivity to H 2 O 2 exposure by analyzing the HyPer-3 fluorescence ratio in the absence or in the presence of the AQP3 inhibitor, DFP00173. ( a ) A representative trace showing the HyPer-3 fluorescence ratio in HCT8 cells stimulated with external 50 μM H 2 O 2 in the absence and the presence of 5 μM DFP00173. ( b ) The histogram shows the normalized HyPer-3 fluorescence ratio. The normalized HyPer-3 fluorescence ratio was measured by calculating the fluorescence ratio at the peak compared to the starting fluorescence values. Data are shown as means ± Standard Error Means (S.E.M.) of five independent experiments and analyzed by paired t test (** p < 0.01 vs. 50 µM H 2 O 2 ).

Article Snippet: Then, the fluorescence emission signal was measured using the FLUOstar Omega Microplate Reader (BMG Labtech, Ortenberg, Germany).

Techniques: Fluorescence

Journal: Antioxidants

Article Title: MOMAST ® Downregulates AQP3 Expression and Function in Human Colon Cells

doi: 10.3390/antiox14010026

Figure Lengend Snippet: Live-cell imaging of changes in HyPer-3 fluorescence in HCT8 cells. ( a ) Representative traces showing the HyPer-3 fluorescence ratio in HCT8 cells treated with MOMAST ® . ( b ) The histogram shows the normalized HyPer-3 fluorescence ratio where the normalized HyPer-3 ratio was calculated by evaluating the fluorescence ratio at the peak compared to the starting fluorescence values. Data are shown as means ± Standard Error Means (S.E.M.) of five independent experiments and analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test (* p < 0.05 vs. 50 µM H 2 O 2 ).

Article Snippet: Then, the fluorescence emission signal was measured using the FLUOstar Omega Microplate Reader (BMG Labtech, Ortenberg, Germany).

Techniques: Live Cell Imaging, Fluorescence

Journal: Toxicology

Article Title: Knock-down of FOXO3, GATA2, NFE2L2 and AHR promotes doxorubicin-induced cardiotoxicity in human cardiomyocytes.

doi: 10.1016/j.tox.2024.153977

Figure Lengend Snippet: Fig. 6. Down-stream molecular effects of TF knock-down in human cardiomyocytes after doxorubicin exposure. A) Human CMs transduced with lentiviral shRNA construct targeting FOXO3, GATA2, NFE2L2 and AHR or LacZ a negative control were exposed to 2 µM DOX for 1 h after which single cells were analyzed for DOX uptake using FACS. Overlay of representative histogram plots for each cell model is shown on the left (>10,000 events per cell model). Quantification of doxorubicin uptake plotted as mean fluorescent intensity per cell model. Data represent mean ±s.d. (n=3), P-values obtained with Mann-Whitney test * P ≤0.05. TF-knockdown cells B and C) Human CMs transduced with lentiviral shRNA construct targeting FOXO3, GATA2, NFE2L2 and AHR or LacZ a negative control were exposed to 1 µM DOX for 24 h. Expression of DDR genes CDKN1A and GADD45A was assessed using RT-qPCR and normalized for HPRT expression. The fold-change per knock-down model is shown relative to shLacZ control for DOX exposed samples. Data represent mean ±s.d. (n=3), P-values obtained with Mann-Whitney test * P ≤0.05. D) For COMET assay lentiviral transduced knock-down cells were exposed for 24 h to 2 µM DOX. Representative images for individual cell models shown upon DMSO or DOX treatment (top panel). Quantification of COMET tail intensity of 3 independent experiments, quantifying 100 cells per condition in each experiment (bottom panel) P-values obtained with One-way Anova, **** P ≤0.0001. E) Representative WB of cleaved caspase 3 and actin as loading control for knock-down models after 24 h of exposure to 2 µM DOX. Quantification of cleaved-caspase 3 expression normalized for actin levels of 3 independent exposures. Data represent mean ±s.d., * P ≤0.05, ** P ≤0.01, **** P ≤0.0001 by Kruskall-Wallis test or One-way Anova (for the COMET-assay).

Article Snippet: After treatment 20 μl of AlamarBlue was added to each well (10 % of total volume) and the plates were incubated at 37 0C for 4 hours before fluorescent emission signal was measured at 590 nm using a CLARIOstar plate reader (BMG Labtech).

Techniques: Knockdown, Transduction, shRNA, Construct, Negative Control, MANN-WHITNEY, Expressing, Quantitative RT-PCR, Control, Single Cell Gel Electrophoresis

Journal: STAR Protocols

Article Title: Protocol to measure contraction, calcium, and action potential in human-induced pluripotent stem cell-derived cardiomyocytes

doi: 10.1016/j.xpro.2021.100859

Figure Lengend Snippet: Imaging-based fluorescence measurement of the action potential (A) The acquisition interface of the line-scan mode. (B) Representative line-scan image of the action potential. (C) Representative fluorescence signals of action potential trace. (D) Example of single action potential trace (shown in the box in panel C) showing the measurement of amplitude and action potential duration (APD).

Article Snippet: Fura-2 AM loaded cells are excited with Lambda DG 4 ultra-high-speed wavelength switching light source (Sutter Instrument, USA) at both 340 and 380 nm at fast-switching mode and the emission fluorescence signals are collected at 510 nm with the iXon Ultra 897 EMCCD camera (Andor Technology, UK).

Techniques: Imaging, Fluorescence