After treatments, cells were washed twice with PBS and then collected in 1 ml of buffer A (PBS/20 mM HEPES, 5 mM d -glucose, pH 7.5) into pre-weighed microfuge tubes. Then, the cells were centrifuged (1,700 g, 5 min, 4 °C), the supernatants were removed and cell pellets were resuspended in 20 vol of buffer A. The cell suspensions were then incubated with fresh Na2S solution (100 μM) prepared in 0.2 M HEPES (pH 7.5) and incubated at 37 °C for 15 min. Thereafter, the cells were centrifuged (3,000 g, 1 min, 4 °C) and 50 μl of the supernatant was mixed with 50 μl of 25 μM 7-Azido-4-Methylcoumarin (AzMC) fluorescent dye (Sigma, 802409) in a 96-well microplate and incubated for 30 min at room temperature. Finally, the samples were analyzed using fluorescence plate reader (Ex: 356 nm, Em: 450 nm). The sulfide concentrations were determined by comparison of the fluorescence intensity to a standard curve prepared using Na2S (0–100 μM).
7 azido 4 methylcoumarin azmc fluorescent dye
Manufactured by Merck Group
7-Azido-4-Methylcoumarin (AzMC) is a fluorescent dye. It exhibits blue-green fluorescence upon excitation.
Automatically generated - may contain errors
2 protocols using 7 azido 4 methylcoumarin azmc fluorescent dye
1
Quantification of Cellular Sulfide Levels
2
Fluorometric Assay for H2S Production
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H2S production was measured using 7-azido-4-methylcoumarin (AzMC) fluorescent dye (Sigma, #802409). The protocol was adapted from Szabo Laboratory with minor modifications (Szabo et al., 2014 (link)). AzMC reaction master mix consisted of 200 mM Tris-HCl pH 8, 20 mM L-cysteine (Sigma, #30089), 1 mM L-homocysteine (Sigma, #69453), 100 μM pyridoxal 5’-phosphate hydrate (Sigma, #P9255), 20 μM AzMC in H2O was prepared and kept on ice. Cells were washed with cool PBS and then harvested in cell lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% v/v IGEPAL CA-630(Sigma-Aldrich-I3021), 1% v/v Triton-X100). Samples were kept on ice for 1 hr and then centrifuged at 20,000 g for 10 min at 4°C before protein quantification by DC protein assay; 400 µg proteins were mixed with 100 μl AzMC reaction master mix and incubated at 37°C in dark for 2 hr. Samples were read at 340 nm excitation and 445 nm emission wavelength using Cytation 3 cell imaging multi-mode reader (BioTek).
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