Mouse brain homogenates were sequentially extracted first in radioimmunoprecipitation assay buffer (RIPA) for the soluble and then in formic acid (FA) for the insoluble protein fraction as previously described (Firuzi et al., 2008 (link); Giannopoulos et al., 2013). Aβ1-40 and Aβ1-42 levels were assayed by a sensitive sandwich ELISA kits (WAKO Chem., Richmond, VA). LTB4 levels were assayed by a colorimetric competitive enzyme immunoassay ELISA kit (ENZO Life Sciences, Farmingdale, NY). Analyses were always performed in duplicate and in a coded fashion.
Colorimetric competitive enzyme immunoassay elisa kit
Manufactured by Enzo Life Sciences
The Colorimetric competitive enzyme immunoassay ELISA kit is a laboratory equipment used for quantitative analysis of specific analytes in a sample. It utilizes the principle of competitive binding to determine the concentration of the target analyte.
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2 protocols using colorimetric competitive enzyme immunoassay elisa kit
1
Amyloid-Beta and LTB4 Quantification
2
Testosterone and ADP/ATP Ratio Quantification
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Testosterone content in serum was determined using a colorimetric competitive enzyme immunoassay ELISA kit (Enzo Life Sciences, Farmingdale, NY) following the protocol according to the manufacturer's instructions. A calibration curve was created using the testosterone standard included in the kit, which has a range from 2000 pg/mL to 7.81 pg/mL. The absorbance was read at 405 Nm using a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA).
The adenosine diphosphate/adenosine triphosphate (ADP/ATP) ratio was determined using single-cell suspensions with a luciferin-luciferase bioluminescent assay kit (Abcam, Cambridge, MA) according to manufacturer's instructions. The ratio was calculated based on a calibration plot made with a standard ATP solution with a range from 1 nM to 10 nM (ATP Disodium Trihydrate, Amresco, Solon, OH). Single-cell suspensions were created using 200 mg of flash-frozen tissues and dissociated with collagenase type I (Stemcell Technologies, Vancouver, Canada) and suspended in phosphate buffered saline (PBS). Luminescence was determined using a Molecular Devices SpectraMax M5 plate reader (Sunnyvale, CA). The data are plotted as a scatterbox using GraphPad Prism to display the average and range of the data points.
The adenosine diphosphate/adenosine triphosphate (ADP/ATP) ratio was determined using single-cell suspensions with a luciferin-luciferase bioluminescent assay kit (Abcam, Cambridge, MA) according to manufacturer's instructions. The ratio was calculated based on a calibration plot made with a standard ATP solution with a range from 1 nM to 10 nM (ATP Disodium Trihydrate, Amresco, Solon, OH). Single-cell suspensions were created using 200 mg of flash-frozen tissues and dissociated with collagenase type I (Stemcell Technologies, Vancouver, Canada) and suspended in phosphate buffered saline (PBS). Luminescence was determined using a Molecular Devices SpectraMax M5 plate reader (Sunnyvale, CA). The data are plotted as a scatterbox using GraphPad Prism to display the average and range of the data points.
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