HPLC measurements of serotonin, and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were performed using HPLC with electrochemical detection (Eicom, HTEC-500, Sapporo, Japan), with a carbon electrode WE-3G (Eicom, Sapporo, Japan), using +650 mV applied potential, and equipped with a reverse-phase column CA-50DS (150 × 2.1 mm, Eicom, Sapporo, Japan), at a flow rate of 200 μL/min. Cells in logarithmic growth phase were trypsinized, washed 2 times with PBS, and immediately frozen and stored at −80 °C in a refrigerator. The cell samples were homogenized in 0.1 M HClO4, centrifuged (10 min, +4 °C; 14,000× g), and filtered using centrifuge filter units (PVDF membrane, pore size 0.22 μm, Millipore, Burlington, MA, USA). The mobile phase contained 100 mM PBS, 0.17 mM disodium ethylenediaminetetraacetate (EDTA), 1.8 mM octane sulfonic acid sodium salt, and 18% (vol/vol) methanol, pH 4.5. All peaks obtained were normalized to the internal standard 3,4-dihydroxybenzylamine, and final values for serotonin and 5-HIAA were counted as ng/mL cell extract of equal number of cells.
Ca 50ds
Manufactured by Eicom
Sourced in Japan
The CA-50DS is a laboratory instrument designed for conductivity and TDS (total dissolved solids) measurements. It features a digital display and provides accurate measurements of conductivity and TDS values in various solutions.
Automatically generated - may contain errors
Lab products found in correlation
Htec 500, by Eicom (5 mentions)
We 3g, by Eicom (4 mentions)
Polyvinylidene fluoride pvdf membrane pore size 0.22 μm, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Polyvinylidene fluoride pvdf membrane, by Merck Group (1 mentions)
5 protocols using ca 50ds
1
HPLC Quantification of Serotonin and 5-HIAA
2
HPLC Analysis of Neurotransmitters
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For HPLC, cell cultures were stabilized at HClO4 (final concentration 0.1 M) and stored at −80 °C until measurements were taken. Sample were thawed, homogenized using ultrasound, filtered in columns (PVDF, Millipore, Tullagreen, Co Cork, Ireland) by centrifugation 14,000× g 1 min., 4 °C before measurement. The supernatant was used for measurement of dopamine (DA), serotonin (5HT), norepinephrine (NA) and their metabolites: homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-indoleacetic acid (5-HIAA) concentrations using the reverse method phase high-performance liquid chromatography with electrochemical detection. The measurement was carried out on an Eicom HTEC-500 chromatograph with WE-3G carbon electrode (Eicom, Kyoto, Japan) at +650 mV potential. Separation was carried out on a reverse phase column CA-50DS (150 × 2.1 mm, Eicom, Kyoto, Japan) with a flow rate of 200 µL/min. Composition mobile phases: per 1 L of solution: 14.9 g NaH2PO4, 1.376 g Na2HPO4, 50 mg EDTA, 400 mg sodium octylsulfonate, methanol 19.5%, pH 4.2.
3
Quantification of Dopamine in Rodent Brain
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The striatum and the frontal cortex (FC) of animals were dissected on ice, frozen in liquid nitrogen, and stored at −80 °C. For analysis, the samples were homogenized in 0.1 M HClO4 (20 μL per mg of tissue) containing 100 ng/mL 3,4-dihydroxybenzylamine (DHBA) as an internal standard. Homogenates were centrifuged for 10 min at 10,000× g (at +4 °C). Supernatants were filtered through centrifuge filter units [polyvinylidene fluoride (PVDF) membrane; pore size 0.22 μm, Millipore, Burlington, MA, USA] and then analyzed for levels of DA, using HPLC with electrochemical detection (Eicom, HTEC-500, Japan) with a carbon electrode WE-3G (Eicom, Tokyo, Japan), using +650 mV applied potential. The separation was carried on a reverse-phase column CA-50DS (150 × 2.1 mm, Eicom, Japan) at 200 μL/min flow rate. The mobile phase contained 100 mM sodium-phosphate buffer, 0.17 mM EDTA, 1.8 mM octanesulfonic acid sodium salt, and 19.5% (vol/vol) methanol, pH 4.31. The volume of injection was 10 μL. All peaks obtained were normalized to internal standard 3,4-dihydroxybenzylamine, and final values were expressed as nanograms per milligram of wet tissue weight.
4
HPLC Analysis of Monoamines in Tissues
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High-performance liquid chromatography (HPLC) measurements of tissue serotonin (5-HT), dopamine (DA), norepinephrine (NE) and their metabolites 5-hydroxyin- doleacetic acid (5-HIAA), 3,4-Dihydroxyphenylacetic acid (DOPAC), and Homovanillic acid (HVA) were performed as described previously (Belov et al., 2019 (link)). Briefly, the striatum, the frontal cortex, the hypothalamus, and the hippocampus were dissected on ice, frozen in liquid nitrogen, and stored at −80°C. The samples for analysis were homogenized in 0.1 M HClO4, centrifuged (10 min, + 4°C; 14,000 × g), and filtered using centrifuge filter units [polyvinylidene fluoride (PVDF) membrane; pore size, 0.22 μm, Millipore, Burlington, MA, United States]. Measurements of monoamines in the tissue samples were performed using HPLC with electrochemical detection (Eicom, HTEC-500, Japan) with a carbon electrode WE-3G (Eicom, Japan) using +650 mV applied potential. The system was equipped with a reverse-phase column CA-50DS (150 × 2.1 mm, Eicom, Japan) at 200 μl/min flow rate. The mobile phase contained 100 mM sodium-phosphate buffer, 0.17 mM EDTA, 1.8 mM octanesulfonic acid sodium salt, and 18% (vol/vol) methanol, pH 4.5. All peaks obtained were normalized to internal standard 3,4-dihydroxybenzylamine, and final values for monoamines and metabolites levels were expressed as nanogram per milligram of wet tissue weight.
5
HPLC Quantification of Brain Serotonin
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HPLC measurements of tissue 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were carried out as described before Belov et al. (2019 (link)). Briefly, the striatum, the frontal cortex, the hypothalamus, and the hippocampus were dissected on an ice, frozen in a liquid nitrogen, and stored at −80°C. The samples for analysis were homogenized in 0.1 M HClO4, centrifuged (10 min, +4°C; 14,000× g) and filtered using centrifuge filter units [polyvinylidene fluoride (PVDF) membrane; pore size, 0.22 μm, Millipore, Burlington, MA, USA]. Measurements of 5-HT and 5-HIAA in the tissue samples were performed using HPLC with electrochemical detection (Eicom, HTEC-500, Japan) with a carbon electrode WE-3G (Eicom, Japan) using +650 mV applied potential. The system was equipped with a reverse-phase column CA-50DS (150 × 2.1 mm, Eicom, Japan) at a flowrate of 200 μl/min. The mobile phase contained 100 mM sodium-phosphate buffer, 0.17 mM EDTA, 1.8 mM octanesulfonic acid sodium salt, and 18% (vol/vol) methanol, pH 4.5. All peaks obtained were normalized to internal standard 3,4-dihydroxybenzylamine, and final values for 5-HT and 5-HIAA were expressed as nanogram per milligram wet tissue weight. The serotonin turnover rate was calculated as a ratio of 5-HIAA/5-HT.
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