The impact of A. absinthium extract on fungal membrane permeability (nucleotide leakage) was determined according to previously published protocol [23 (link), 24 (link)] with some modifications and compared to untreated yeast cells. The culture of C. albicans 475/15 incubated overnight at 37°C was washed twice and resuspended in 10 mM PBS (pH 7.4), reaching the final density of 108 CFU/mL. Strain was incubated with the extract at the MIC for 30 min; C. albicans incubated with 10 mM PBS (pH 7.4) was used as control. After incubation, cell suspensions were centrifuged at 10,000 g for 10 min and supernatant absorbance measured at 260 nm and 280 nm with Agilent/HP 8453 UV-Visible Spectrophotometer (Agilent Technologies, USA) at room temperature (25°C).
Agilent hp 8453 uv visible spectrophotometer
Manufactured by Agilent Technologies
Sourced in United States
The Agilent/HP 8453 UV-Visible Spectrophotometer is a laboratory instrument used to measure the absorption or transmission of light in the ultraviolet and visible spectrum. It is designed to analyze the chemical composition and properties of various samples.
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7 protocols using agilent hp 8453 uv visible spectrophotometer
1
Fungal Membrane Permeability Analysis
2
Measuring Candida albicans Oxidative Stress
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These studies were carried out with suspensions of C. albicans 475/15, supplemented with MICs, ½ MICs and ¼ MICs of apigenin-7-O-glucoside and apigenin. For the nitro blue tetrazolium (NBT) reaction (Páez et al., 2010[24 (link)]) 0.4 mL of yeast suspension treated overnight with apigenin-7-O-glucoside and apigenin (OD600 nm 0.8) and 0.5 mL of 1 mg/mL NBT were incubated for 30 min at 37 °C. Then, 0.1 mL of 0.1 M HCl was added and the tubes were centrifuged at 2500 g for 10 min, with the blue color of supernatants being measured at 575 nm (ROS extracellular). The separated pellets were treated with 0.6 mL dimethyl sulfoxide (DMSO) to extract the reduced NBT, and finally, 0.8 mL phosphate saline buffer was added and OD575 nm was determined (ROS intracellular) using Agilent/HP 8453 UV-Visible Spectrophotometer (Agilent Technologies, USA).
3
Intracellular ROS Levels in C. albicans
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The impact of compounds on intracellular levels of ROS was determined according to Paez et al. [55 (link)]. C. albicans 475/15 was incubated with MICs of compounds overnight at 37 °C. The suspension of C. albicans treated cells (0.4 mL) was further incubated with 0.5 mL of nitro blue tetrazolium (1 mg/mL) at 37 °C for 30 min. After the addition of 0.1 mL 0.1 M HCl, tubes were centrifuged at 2500× g for 10 min. The pellets were treated with 0.6 mL dimethyl sulfoxide and 0.8 mL phosphate saline buffer and absorbance was recorded at 575 nm (Agilent/HP 8453 UV-Visible Spectrophotometer; Agilent Technologies, Santa Clara, CA, USA).
4
Rutin's Effect on Bacterial Membrane Permeability
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The effect of rutin on membrane permeability (nucleotide and protein leakage) was evaluated as described earlier with some modifications [76 (link)]. The culture of P. aeruginosa IBRS P001 and MRSA IBRS MRSA 011 was incubated overnight at 37 °C, washed, and suspended in 10 mM PBS in order to achieve the final density of 108 CFU/mL. Bacteria were incubated with rutin at a concentration equal to MIC for time intervals 0, 30, 60, and 90 min. Bacteria incubated with PBS were used as control. After incubation the mixture was filtered through a 0.22 μm pore size filter to remove the yeast cells. The optical density of the filtrate was measured at 260 and 280 nm with an Agilent/HP 8453 UV-Visible Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) at room temperature (25 °C).
5
Astragalin Impacts Candida albicans Membrane
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The impact of astragalin on membrane permeability of C. albicans 475/15 was determined according to Tang et al. 2008[27 ], with some modifications. C. albicans 475/15 was incubated overnight at 37 °C, washed and suspended in 10 mM PBS (pH 7.4). Density of cells was adapted to 108 CFU/mL. C. albicans was incubated with astragalin at 1½ MICs for: 0, 15, 30, 45 and 60 min; astragalin dissolved in PBS was used as blank. The mixture was filtered through 0.22 μm pore size filter and optical density was recorded at 260 nm and 280 nm with Agilent/HP 8453 UV-Visible Spectrophotometer Agilent Technologies, USA, at room temperature.
6
Astragalin's Impact on Intracellular ROS in C. albicans
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The impact of astragalin on levels of intracellular ROS was determined according to the method described by Paez et al. (2010[15 (link)]). C. albicans 475/15 was incubated with MIC of astragalin overnight at 37 ºC; 0.5 mL of 1 mg/mL nitro blue tetrazolium (NBT) was added and incubation was continued at 37 ºC for 30 min. After addition of 0.1 mL 0.1 M HCl tubes were centrifuged at 2500 g for 10 min. Dimethyl sulfoxide (0.6 mL) and phosphate saline buffer (0.8 mL) was added to the pellet and absorbance was recorded at 575 nm using Agilent/HP 8453 UV-Visible Spectrophotometer (Agilent Technologies, USA).
7
Apigenin-7-O-glucoside Membrane Permeability
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The effect of apigenin-7-O-glucoside on membrane permeability (nucleotide leakage) was evaluated according to Tang et al. (2008[31 ]) with some modifications and compared to effect of apigenin. The culture of C. albicans 475/15 incubated overnight at 37 °C was washed and resuspended in 10 mM PBS (pH 7.4), reaching the final density of 108 CFU/ ml. Strain was incubated with the target molecules at the 1½ MICs for different time intervals: 0, 15, 30, 45 and 60 min; C. albicans incubated with 10 mM PBS (pH 7.4) was used as control. The mixture was filtered through 0.22 μm pore size filter to remove the yeast cells. The optical density of the filtrate was measured at 260 nm and 280 nm with Agilent/HP 8453 UV-Visible Spectrophotometer Agilent Technologies, USA) at room temperature (25 °C).
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