Serum was stored at −80 °C until analysis, then thawed. Measurements were conducted in duplicate using commercial ELISA kits: melatonin (Cloud-Clone Corp., Houston, TX, USA); corticosterone, TNFα, IgM (Abcam, Cambridge, MA, USA); IgG (Abnova, Neihu District, Taipei City, Taiwan). Immunoassay results were read with a fluorescence multi-detection reader (Bio-Tek Instruments, Winooski, VT, USA) at the indicated wavelength. Assay concentrations were quantitated using GraphPad PRISM software (GraphPad Software, La Jolla, CA, USA). A nonlinear regression analysis was used to derive an equation to predict the concentration in unknown samples.
Fluorescence multi detection reader
Manufactured by Agilent Technologies
Sourced in United States
The Fluorescence multi-detection reader is a versatile laboratory instrument used to measure fluorescence intensity in various sample types. It is designed to provide accurate and reliable fluorescence detection across a range of applications.
Automatically generated - may contain errors
8 protocols using fluorescence multi detection reader
1
Serum Biomarker Quantification Protocol
2
Intracellular ROS Measurement by DCF-DA Assay
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Intracellular ROS levels were measured using a DCF-DA assay as previously described [51 (link)]. Cells were seeded in 96-well dark plates (1 × 104 cells/well). After starvation with serum-free medium, the cells were treated vehicle or WGP in the presence of absence of 100 mM ethanol for 24 h and then washed twice with ice-cold phosphate-buffered saline (PBS). The cells were incubated with 20 µM DCF-DA for 1 h at 37 °C in darkness. After washing with PBS twice, the detection was performed using a fluorescence multi-detection reader (BioTek Instruments, Winooski, VT, USA) with excitation at 485 nm and emission at 535 nm.
3
ROS Generation Assay with AuNPs
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ROS generation was measured according to the method described earlier, using 2′,7′-dichlorofluorescein diacetate (DCFDA) [43 ]. Bacterial cells (106 CFU/mL) were treated at the required temperature for 2 h, with AuNPs and without AuNPs. After incubation, the cells were centrifuged at 4 °C for 30 min at 300 × g, and each supernatant was treated with 100 μM DCFDA for 1 h. The ROS formed in the sample were detected at 485/20 nm of fluorescence excitation wavelength and 528/20 nm of emission wavelength using a fluorescence multi-detection reader (Bio Tek, Winooski, VT, USA).
4
Quantifying Bacterial ROS Generation
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ROS generation was measured according to the previously described method using DCFDA [23 (link),56 (link)]. Bacterial cells (106 CFUs/mL) were treated with or without AgNPs at the required temperature for 12 h. After incubation, cells were centrifuged at 4 °C for 30 min at 300× g, after which the supernatant was treated with 100 μM DCFDA for 1 h. The amount of ROS produced in the sample was detected at the excitation wavelength of 485/20 nm and emission wavelength of 528/20 nm using a fluorescence multi-detection reader (BIOTEK, Winooski, VT, USA).
5
ROS Generation Measurement Using DCFDA
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ROS generation was measured according to the previously described method using 2′,7′-dichlorofluorescein diacetate (DCFDA) [52 ,95 (link)]. Bacterial cells (106 CFU/mL) were treated with or without AgNPs at the required temperature for 12 h. After incubation, cells were centrifuged at 4 °C for 30 min at 300× g, after which the supernatant was treated with 100 μM DCFDA for 1 h. The amount of ROS produced in the sample was detected at the excitation wavelength of 485/20 nm of fluorescence excitation and emission wavelength of 528/20 nm using a fluorescence multi-detection reader (BIOTEK, Winooski, VT, USA).
6
Evaluating Nanoparticle-Induced ROS in E.coli
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The ROS formation in the control and experimental group was measured by 2′, 7′- dichlorofluorescein diacetate (DCFDA) method. The test bacteria E.coli was treated with nanoparticle samples at 37 °C for 3 h. The bacteria without nanoparticles treatment was considered as a control. After, the control and experimental sample was centrifuged at 10,000 rpm for 10 min. The collected supernatant was incubated with 100 µM of DCFDA for 1 h. The production of ROS in the samples was measured at 485/20 nm of fluorescence excitation wavelength and 528/20 nm of emission wavelength using Fluorescence Multi-Detection Reader (BIOTEK, USA).
7
Quantification of Serum TNF-α Levels
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Serum was stored at -80°C until analysis, then thawed, and run in duplicate. TNF-α levels were quantified using an ELISA kit (eBioscience, San Diego, CA, USA). Immunoassay results were then read with a fluorescence multi-detection reader (Bio-Tek Instruments, Winooski, VT, USA) at 620 and 450 nm. The concentrations were quantitated using the GraphPad PRISM software (ver. 5.0; GraphPad Software, La Jolla, CA, USA). A non-linear regression analysis was used to derive an equation to predict the concentration in unknown samples.
8
Quantification of Cerebrospinal Fluid Melatonin
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Cerebrospinal fluid (CSF) samples were collected from the cisterna magna at ZT13-ZT15. Since repeated CSF collection might alter the physiological state of the animals, CSF was obtained right before the sacrifice. Collected CSF samples were centrifuged at 2000× g for 10 min and stored at −80 °C until further analysis. The samples were thawed and run in at least triplicate. The melatonin levels were quantified using commercially available enzyme-linked immunosorbent assay kits (Cloud-Clone Corp., Houston, TX, USA). Immunoassay was performed using the Fluorescence Multi-Detection Reader (BIOTEK, Winooski, VT, USA) at an absorbance of 450 nm. The concentration of melatonin was quantified using the GraphPad PRISM 5.0 program (GraphPad Software, La Jolla, CA, USA). A nonlinear regression analysis was used to derive an equation to predict the concentration of the unknown samples.
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