Brains from 6 adult male wildtype and 9 adult male raphe KO mice were rapidly removed and single coronal slices between bregma −0.9 and −3.0 were cut and placed on a rubber stopper. Then, dorsolateral neocortex, amygdala, hippocampus and hypothalamus were dissected from the slices with a scalpel and forceps. The tissue samples were homogenized in 100 µl of 0.1 N HClO4 and centrifuged at 12,200 x g for 15 min. Concentrations of 5-HT and its acid metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were quantified in the supernatant using high-pressure liquid chromatography with electrochemical detection (HPLC-ECD). Aliquots were injected onto an HPLC column linked to a coulometric detector (ESA Model Coulochem III, Dionex Corp., Chelmsford, MA, USA). Mobile phase consisting of 50 mM sodium phosphate monobasic, 250 mM Na2EDTA, 0.03% sodium octanesulfonic acid and 25% methanol (pH 2.75) was recirculated at 0.9 ml/min. Data were acquired by an Empower software system (Waters Corp., Milford, MA, USA), where peak heights of unknowns were compared to standards. The lower limit of assay sensitivity (3 x baseline noise) was 1 pg/20 ml sample. One KO hippocampal sample was contaminated by striatum and not used and one WT hypothalamic sample was lost.
Empower software system
Manufactured by Waters Corporation
Sourced in United States
Empower software system is a data acquisition and management software used for analytical instrumentation. It provides a platform for collecting, analyzing, and reporting data generated from various laboratory instruments.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using empower software system
1
Quantification of 5-HT and 5-HIAA in Mouse Brain
2
Quantification of Monoamines in Striatal Tissue
3
Rat Liver 5α-Reductase Enzyme Assay
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The 5 α-reductase enzyme from rat liver microsomes was incubated with 400 μL of phosphate buffer (pH 6.5) in the Intact group, and 200 μL of phosphate buffer in the normal group (negative control). Testosterone was used as a substrate for 5α-reductase at a volume of 50 μL (100 μg/mL). The other samples were incubated with 200 μL (1 mM) of finasteride, which was considered the positive control group (finasteride), or 200 μL (1 mM) of compounds (1 and 2 ). Finally, 20 μL of NADPH (0.8 mg/mL) was added. Microsomal enzymes (5α-reductase) isolated from rat livers were added to the samples from all groups, except for the intact group. The reaction was terminated by adding 0.5 mL of dichloromethane for all group. The amount of testosterone in the samples was measured by HPLC. The injection volume was 20 μL and elution was performed at a flow rate of 1 mL/min, using a binary gradient of water (A) and acetonitrile (ACN) (B). The quantification wavelength of these chromatograms was set at 242 nm, which was optimized for testosterone. The data were integrated using the Empower software system (Waters, Coastal, CT, USA).
4
Rat Liver 5α-Reductase Assay
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5α-Reductase from rat liver microsomes was incubated with 400 μL phosphate buffer (pH 6.5) on Intact group and 200 μL phosphate buffer on Normal group (negative control), 50 μL testosterone as substrate (100 μg/mL), 200 μL (1 mM) finasteride on positive control group (finasteride) or 200 μL (1 mM) of compounds (1 –7 ), and 20 μL NADPH (0.8 mg/mL). Microsome enzyme (5α-reductase) isolated from rat liver was added to all groups except for the intact group with 200 μL. The reaction was terminated by 0.5 mL dichloromethane was added for every group. The amount of testosterone as substrate for 5α-reductase was measured by HPLC. The injection volume was 20 μL and elution was performed at a flow rate of 1 mL/min using a binary gradient of H2O (A) and acetonitrile (ACN) (B). The quantification wavelength of these chromatograms was set at 242 nm, which was optimized for testosterone. The data were integrated using the Empower software system (Waters, coastal, CT, USA).
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