C. difficile toxins A and B were quantified separately by C. difficile toxin A or B quanti kit (tgcBiomics GmbH, Germany) using sandwich enzyme-linked immunoassay (ELISA) [8 (link)]. Stool samples (50 μL if liquid/semiliquid or 50 mg if solid) were diluted at a ratio of 1:10 by dilution buffer (450 μL) provided with the kit. Samples were then homogenized with disposal pipetting suction and ejection or vortexing followed by centrifugation at 2500×g for 5 min. One hundred μL of clean supernatant was pipetted and poured into the microtiter plate well for toxin A or B analysis. Plates were read by Synergy HT microplate reader (BioTek Instruments, Inc., Winooski, VT) and measured optical density was analysed into respective concentration values by Gen5 software version1.09 (BioTek Instruments, Inc., Winooski, VT). The lower and upper limits of detection of the assay were 0.3 and 80 ng/mL, respectively, for both toxin A and B.
Gen5 software version 1.09
Manufactured by Agilent Technologies
Sourced in United States
Gen5 software (version 1.09) is a data analysis platform developed by Agilent Technologies. It provides tools for the management and analysis of data generated from various laboratory equipment and instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using gen5 software version 1.09
1
Quantifying C. difficile Toxins A and B
2
Quantifying Cellular Lactate Dehydrogenase Activity
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The lactate dehydrogenase (LDH) technique was followed as described by Alarifi et al (2018). Briefly, 50 μl of culture media was withdrawn from experimental plates and mixed with 50 μl lithium lactate solution of 50 mM, 50 μl tris solution of 200 mM, and 50 μl of NAD solution (a mixture of nitrotetrazolium violet, phenazine metosulphate, and nicotinamide dinucleotide; Sigma Aldrich, Merck KGaA, Darmstadt, Germany) were added to a new 96 wells plate. After 15 min, the absorbance was taken at 490 nm and the background absorbance was taken at 690 nm using a microplate reader with Gen5 software (version 1.09) (BioTek Instruments, Winooski, VT, USA).
3
Quantifying Oxidative Stress in Liver Cells
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Generation of ROS levels in liver cells was determined by Ali et al,9
due to exposure to La2O3 NPs (0, 10, 20, 50, 100, and 300 μg/ml) for 48 h. The cells (3×104) were seeded in a black bottom culture plate (96 well) and incubated in a CO2 incubator at 37°C for 24 h for attachment. After exposure to La2O3 NPs, the culture plates were incubated for 48 h. After incubation, 10 μM DCFH-DA was added per well for 40 min at 37°C. After incubation, the plate was washed, and fluorescence intensity was measured at 485 nm excitation and 520 nm emissions using by a microplate reader with Gen5 software (version 1.09) (Bio-Tek Instruments, Winooski, VT, USA). Data were represented as a percent of fluorescence intensity in comparison to the control wells. A separate set of experiments were carried out to assess for generation of ROS through the qualitative analysis method.9
due to exposure to La2O3 NPs (0, 10, 20, 50, 100, and 300 μg/ml) for 48 h. The cells (3×104) were seeded in a black bottom culture plate (96 well) and incubated in a CO2 incubator at 37°C for 24 h for attachment. After exposure to La2O3 NPs, the culture plates were incubated for 48 h. After incubation, 10 μM DCFH-DA was added per well for 40 min at 37°C. After incubation, the plate was washed, and fluorescence intensity was measured at 485 nm excitation and 520 nm emissions using by a microplate reader with Gen5 software (version 1.09) (Bio-Tek Instruments, Winooski, VT, USA). Data were represented as a percent of fluorescence intensity in comparison to the control wells. A separate set of experiments were carried out to assess for generation of ROS through the qualitative analysis method.9
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