Lysates of cultured cells were prepared by mixing with lysis buffer (100 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, pH 7.4, and protease inhibitors (Roche, Basel, Switzerland). Total soluble protein was quantitated by bicinchoninic acid protein assay. The reactions were performed with 2 μg of purified HO-1 protein or 10 μL (20-100 μg total protein) of the cell lysates in 100 mM Tris-HCl with 15 μM hemin, 0.8 mM NADPH, 1 mM MgCl2, 0.8 mM glucose 6-phosphate, 300 μM BSA, 1 U glucose-6-phosphate dehydrogenase (Sigma), and 3 μL of Biliverdin reductase (Sigma). The reaction samples were mixed and 200 μL of the mixture was transferred into quartz micro cuvettes (3 × 3 × 40 mm). The fluorescence was detected in a fluorescence spectrofluorometer (Photon Technology International, New Jersey) at 37°C with excitation at 441 nm and emission at 528 nm, using a gain value of 140. Detection of bilirubin emission was performed in real-time kinetics with resolution of 1 sec. A specific HO-1 inhibitor tin Protoporphoryrin IX dichloride (Santa Cruz Biotechnology, Inc., TX) was used as a positive control. Every measurement of each wavelength and time point was detected five times and the arithmetic mean with standard deviation (SD) was calculated.
Fluorescence spectrofluorometer
Manufactured by Horiba
Sourced in Canada, Japan
The Fluorescence Spectrofluorometer is a laboratory instrument used to measure the fluorescence properties of samples. It excites the sample with light and detects the resulting fluorescence emission, providing information about the sample's molecular structure and composition.
Automatically generated - may contain errors
5 protocols using fluorescence spectrofluorometer
1
Heme Oxygenase-1 Activity Assay
2
Synthesis and Characterization of Fluorescent Silica Nanoparticles
3
Synthesis and Characterization of Fluorescent Silica Nanoparticles
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Fluorescent silica nanoparticles (C′ dots) of different size, encapsulating the organic dye, Cy5, were synthesized in water as previously described30 (link). αMSH peptides were conjugated to maleimido-terminated heterobifunctional polyethylene glycol silane (mal-PEG-silane) via its N-terminal acetylated cysteine thiol to form αMSH-PEG-silane. Conjugates were attached to the particle surface in the PEGylation step as described previously10 (link),30 (link) to generate αMSH functionalized C′ dots, or αMSH-PEG-C′ dots. Synthesized particle samples were dialyzed in water and purified by gel permeation chromatography (GPC, Bio-Rad Laboratories, Inc, Hercules, California) prior to further characterization10 (link). Absorption and emission spectral profiles for the encapsulated and native Cy5 dye were obtained using a Varian Cary 5000 spectrophotometer (Varian, Palo Alto, CA) and a fluorescence spectrofluorometer (Photon Technology International, Inc, Birmington, NJ). Hydrodynamic radius, brightness, and concentration of αMSH-PEG-C′ dots, as against free Cy5 dye, were determined using a homebuilt fluorescence correlation spectroscopy (FCS) set up configured with a solid-state 633-nm excitation10 (link).
4
Mitochondrial Membrane Potential Assay
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The Mitochondrial membrane potential was measured using the JC1 Mitochondrial Membrane Potential Assay Kit (Mitosciences, Abcam, Cambridge, UK) following the manufacturer’s instructions. Briefly, after the treatment, cells were washed once with PBS and then incubated with JC-1 (10 μM). The cells were then incubated at 37°C for 10 min. After washing, the cells were analyzed on a fluorescence spectrofluorometer (Horiba, Japan) at 519 nm excitation and 590 nm emission. All the experiments were done in triplicate and the results were as Mean ± SE.
5
Measuring Intracellular Reactive Oxygen Species
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Intracellular reactive oxygen species (ROS) levels were measured using the fluorescent dye DCFDA (2′,7′-dichlorofluoresceindiacetate). After treatment, cells were washed once with PBS and then loaded with 5 μM DCFDA in PBS with 4% FBS. The cells were incubated at 37°C for 30 min in the dark and subsequently washed with PBS, and centrifuged at 12,000 g for 10 min at 37°C. After washing, the cells were analyzed on a fluorescence spectrofluorometer (Horiba, Japan) at 488 nm excitation and 519 nm emission as described (Lee, Pecinova et al., 2010 (link)). All the experiments were done in triplicate and the results were as Mean ± SE.
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