Cell Viability, Metabolism Assays and Biochemicals

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Figure 1. (a) Fluorescent responses of NP3 (5 μM) toward various analytes (10 μM). Data shown represent fluorescent intensity at 470nm, 30 min after addition of the analytes. (b) ONOO− (final 10 μM) was quickly injected into a solution of NP3 (final 5μM), and the fluorescent intensity at 470 nm was plotted against time. (c) Fluorescence enhancement of NP3 (5μM) at 470 nm as a function of ONOO− (0−10 μM) after 15 min of reaction. All data acquired in PBS (10 mM, pH 7.4) with excitation at 375 nm.Figure 2. (a) Time-lapse images taken from living EA.hy926 endothelial cells. Cells were preincubated with NP3 (5.0 μM), followed by stimulation with or without SIN-1 (0.5 mM). (b) Dynamic changes of NP3 fluorescence after SIN-1 (0.5 mM) treatment in panel a. (c) Effects of ONOO− scavengers uric acid (100 μM) and FeTTPS (1 μM) on changes in NP3 fluorescence in endothelial cells in the presence of ONOO− (60 μM). PI (red) stains nuclei. NP3 fluorescence was collected at 420−480 nm with λex 405 nm.
Figure 1. Propidium iodide excitation and emission spectra.Figure 2. HL-60 cells were treated with a drug, then stained with FLICA® FAM-VAD-FMK poly-caspase inhibitor reagent (green) (Cat. 92) and propidium iodide (red) (PI). Scanning laser cytometer analysis. Four populations of cells were detected: (A) Unstained live cells, (B) red necrotic cells (PI), (C) green and red late apoptotic cells (FAM-FLICA® plus PI), and (D) green early apoptotic cells (FAM-FLICA®). Data courtesy of Dr. Z. Darzynkiewicz, Brander Cancer Center, NY.
Figure 1. Assay principle: a non‐fluorescent detection reagent is oxidized in the presence of hydrogen peroxide and MPO to produce its fluorescent analog.Figure 2. A MPO standard curve was prepared and run, as described in the protocol, in the absence (A) or presence (B) of 20 mM catalase inhibitor. MPO standard and reaction mix were added to a 96-well black plate and incubated at room temperature in the dark for 30 minutes. The wells were read using Ex: 530nm and Em: 590nm. There is an approximately 50% reduction in signal in the presence of 20mM catalase inhibitor (note the different scales on the x-axis).

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