We collected the cell culture supernatant from 35 days old organoids 40 hr after feeding and measured its dopamine content using a Dopamine ELISA Kit (Abnova) according to the manufacturer’s instructions. Measurements were performed in duplicates and the sample concentrations were calculated based on a standard curve.
Dopamine elisa kit
Manufactured by Abnova
Sourced in United States
The Dopamine ELISA Kit is a laboratory equipment product designed for the quantitative measurement of dopamine levels in various sample types, such as serum, plasma, urine, and cell culture supernatants. The kit utilizes the enzyme-linked immunosorbent assay (ELISA) technique to detect and quantify the presence of dopamine in the samples.
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17 protocols using dopamine elisa kit
1
Dopamine Quantification in Organoid Culture
2
Quantitative Dopamine Measurement in Conditioned Media
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Conditioned medium from fifty thousand cells cultured for 3 days was collected for dopamine assay using Dopamine ELISA Kit (CAT#KA1887, Abnova). Following the manufacturer’s instruction, samples were processed through extraction, acylation, and enzymatic assay. The extraction step was performed from 10 μl of standards and 300 μl of samples mixed in the extraction plate following by addition of 50 μl of assay buffer and 50 μl of extraction buffer. After shaking incubation for 30 min, the plate was washed twice with washing buffer provided, followed by drying. The acylation step was continued by adding 50 μl of acylation buffer and 25 μl acylation reagent into the extraction plate following 15 min incubation time. The plate was then washed and dried before incubating in 175 μl of hydrochloric acid for 10 min on shaker. The final enzymatic assay was continued with 25 μl of enzyme solution, 25 μl of the extracted standards and 50 μl of the extracted sample following the protocol. Absorbance was measured within 10 min using the microplate reader under the wavelength of 450 nm.
3
Dopamine ELISA Protocol for Mice
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A dopamine ELISA kit (#KA1887, Abnova, Taiwan) was used to evaluate dopamine concentration in the NAcc of the mice. The protocol was conducted according to the manufacturer's instructions provided. Briefly, the standards, controls and samples were subjected to extraction and acylation techniques. The enzyme solution, standards, controls, samples and HCl were added to 96‐well microtitre plates and incubated for 30 min at room temperature after extraction. Next, dopamine antiserum was added and incubated for 2 h at room temperature (20–25°C). Afterward, the samples were discarded and washed three times with a washing buffer. The plates were incubated with the enzyme conjugate for 30 min and washed three times. Plates were then incubated with 3,3′,5,5′‐tetramethylbenzidine substrate for 25 min in the dark. Finally, a stop solution was added to the plate, and an EMax Plus microplate reader (Molecular Devices, San Jose, CA, USA) was used to measure the absorbance at 450 nm. All measurements were performed in duplicates.
4
Monkey and Rat CSF Sampling and Dopamine Quantification
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The cerebrospinal fluid (CSF) of monkeys was sampled by lumbar puncture in L3-4 interspace when the monkeys were under anesthetized by intramuscular injection of atropine (0.05 mg/kg) and ketamine (10 mg/kg). The CSF was protected from light during the sampling process, then the CSF sample (0.8 ml) was centrifuged at 8000 × g for 15 min at 4 °C and immediately frozen at −80 °C until analysis. CSF of rats samples is taken from the cisterna magna using a method as described previously77 . Dopamine concentration of CSF assayed by dopamine ELISA Kit (Abnova, Catalog Number KA1887).
5
Serum Dopamine and Serotonin Measurement
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For serum dopamine level measurement, 200 μL of serum samples were mixed 100 μL of 1 × phosphate-buffered saline (PBS) to adjust the dopamine extraction reaction volume, and subjected to the extraction procedures provided in dopamine ELISA kit (Abnova, Taiwan). For serum serotonin measurement, serum samples were diluted at 1:5 and 1:10 ratios and used with serotonin ELISA kit (Abcam, USA). Competitive ELISA experiments were performed following the manufacturer's instructions.
6
Dopamine Quantification in Fly Heads
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After the 7-day treatment, 50 flies per vial, n = 5, were frozen at −20 °C and their heads were separated from the bodies using forceps and sharp blade/cutter. Then, fly heads were homogenized in 0.1 M Perchloric acid and centrifuged using an Eppendorf Legend Micro 17R Centrifuge (fixed angle rotor) at 12000×g for 30 min at 4 °C. The supernatants were carefully separated from the pellets and stored at −20 °C until used within 24 h. Dopamine ELISA kit (Abnova, Taiwan) was thereafter used to determine the level of dopamine in the supernatants by following the manufacturer’s procedure. Spectramax Plus 384 Microplate Reader (Molecular Devices) was used to measure the absorbance at wavelength of 450 nm.
7
Dopamine and GLP-1 Signaling Pathways
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Salts and organic solvents used in solution preparations were purchased from Fisher Scientific (Leicestershire, UK), Sigma Chemicals (St. Louis, MO, USA, EUA), or Merck (Darmstad, Germany), with the highest grade of purity commercially available. It used antibodies to analyze D1R, D2R, DARPP32 (ab81296, ab85367, and ab40801 respectively, Abcam, UK), and tyrosine hydroxylase (TH), (T1299, Sigma Aldrich, St. Louis, MO, USA) at a 1:1000 dilution. Moreover, antibodies against GLP-1 and GLP-1R and against the phosphorylated form of insulin receptor were used (ab22625, ab218532, and InsR-Tyr972, ab5678 respectively, Abcam, UK) as well, against phosphorylated AMPK form (Thr172, 2535S, Cell Signalling Technology, Danvers, MA, USA, EUA). Calnexin was used as loading control (AB0037, Sicgen, Cantanhede, Portugal). Plasma dopamine levels and plasma GLP-1 levels were assessed through the Dopamine ELISA Kit, (Abnova, Taiwan) and Rat GLP1/Glucagon-like Peptide 1 ELISA Kit, (LifeSpan BioScience, Inc., Washington, DC, USA) respectively.
8
Quantifying Dopamine Release in Neurons
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The obtained dopaminergic neurons (at day 28) were transferred onto the Matrigel-coated 96-well plates, then washed twice with a low KCl solution (phenol-free, Ca2+Mg2+-free Hank’s balanced salt solution (HBSS; Wako Pure Chemicals Industries)) and incubated for 15 min. The medium was subsequently replaced with a high KCl solution (56 mM) for 15 min. The solution was collected, and the concentration of dopamine was determined by a commercial dopamine ELISA kit (Abnova, Taipei, Taiwan).
9
Measuring Neurotransmitters in HFD Mice
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Pancreatic lipase activity was measured as previously described (36) . In brief, FGT was dissolved in distilled water (as a negative control) or in 50
For neurotransmitter measurements, 6-week-old male C57/ BL6 mice were purchased from the Central Laboratory Animal Inc. and adapted for 1 week. After adaptation, mice were fed a 45 % HFD. During the administration of a HFD, FGT (500 mg/kg body weight) or water (as a vehicle) was orally administered for 8 weeks. After FGT administration, mice were fasted overnight, and blood samples were collected and centrifuged (4°C, 3000 rpm, 5 min). Supernatants were transferred to new microcentrifuge tubes. Plasma levels of dopamine, norepinephrine and serotonin were measured using a dopamine ELISA kit (Abnova), norepinephrine ELISA kit (LifeSpan Biosciences) and serotonin ELISA kit (Abcam), respectively, following each manufacturer's instructions. White adipose tissue (WAT) and liver tissue were separated and stored at -80°C for further use. All animal experiments were approved by the Amorepacific Institutional Animal Care and Use Committee (AP11-FR008) and adhered to the OECD guidelines.
For neurotransmitter measurements, 6-week-old male C57/ BL6 mice were purchased from the Central Laboratory Animal Inc. and adapted for 1 week. After adaptation, mice were fed a 45 % HFD. During the administration of a HFD, FGT (500 mg/kg body weight) or water (as a vehicle) was orally administered for 8 weeks. After FGT administration, mice were fasted overnight, and blood samples were collected and centrifuged (4°C, 3000 rpm, 5 min). Supernatants were transferred to new microcentrifuge tubes. Plasma levels of dopamine, norepinephrine and serotonin were measured using a dopamine ELISA kit (Abnova), norepinephrine ELISA kit (LifeSpan Biosciences) and serotonin ELISA kit (Abcam), respectively, following each manufacturer's instructions. White adipose tissue (WAT) and liver tissue were separated and stored at -80°C for further use. All animal experiments were approved by the Amorepacific Institutional Animal Care and Use Committee (AP11-FR008) and adhered to the OECD guidelines.
10
Quantification of Striatal Dopamine
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For the analysis of dopamine content in the striatum, dissected striatal tissues were homogenized in 0.01 N HCl containing 1 mM EDTA and 4 mM sodium metabisulfite and centrifuged at 13,000 rpm for 20 min. The concentration of dopamine in the supernatant was determined using a dopamine ELISA Kit (Abnova, Walnut, CA, USA), according to the manufacturer’s instructions. Briefly, dopamine in the dialysate was extracted using a cis-diol-specific gel, acylated, and derivatized enzymatically. The optical density was determined at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Dopamine levels were normalized to the weight of the wet tissue.
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