Blood samples were collected after the mice were sacrificed. The fresh blood was kept standing for 60 min at 37 °C. Serum was obtained by centrifugation at 3000 rpm for 15 min and then stored at − 80 °C until use. Concentrations of cytokines including IL-4, IL-6, IL-10, CXCL1/KC, and TNF-α in serum were determined using a magnetic Luminex screening assay according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN) on a Bio-Plex 200 analyzer (Bio-Rad, Hercules, CA). In addition, serum corticosterone level was measured using the corticosterone (Abnova, Taipei, Taiwan) and serotonin (Abcam, Cambridge, MA, USA) enzyme-linked immunosorbent assay kits, respectively, according to the manufacturer’s instructions.
Serotonin
Manufactured by Abcam
Sourced in United States
Serotonin is a chemical compound that functions as a neurotransmitter in the human body. It plays a role in various physiological processes, including mood regulation, sleep, and appetite.
Automatically generated - may contain errors
Lab products found in correlation
Bio plex 200 analyzer, by Bio-Rad (1 mentions)
Corticosterone, by Abnova (1 mentions)
Luminex screening assay, by R&D Systems (1 mentions)
Brain derived neurotrophic factor (bdnf), by Abcam (1 mentions)
Glass capillary tubes, by Corning (1 mentions)
Substance p, by R&D Systems (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
βiii tubulin, by Fortrea (1 mentions)
7 protocols using serotonin
1
Cytokine and Hormone Profiling in Mice
2
Plasma biomarkers quantification
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Plasma samples from heparinized blood were separated and used to determine the plasma levels of the study markers. Enzyme-linked immunosorbent assay (ELISA) was conducted using commercially available kits for the measurement of plasma dopamine (MyBioSource, USA), serotonin (Abcam, UK), and BDNF (Abcam, UK). Technical procedures were according to the manufacturer’s instructions.
3
Quantifying Tear Neuromediators Using ELISA
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Tear samples were carefully obtained from the inferior tear meniscus of the right eye using glass capillary tubes (Corning, Inc., Corning, NY, USA) to avoid touching the ocular surfaces after examination of their clinical parameters. Two hundred microliters of tear samples were obtained and diluted with phosphate-buffered saline (1:4). The samples were placed in microtubes and stored at −70℃ until further examination.18 (link)
A specific enzyme-linked immunosorbent assay was performed on extracted proteins to quantify the concentrations of neuromediators in the tear samples of subjects following manufacturer instructions. Tear samples were centrifuged at 3000 g for 10 min and filtered through a 0.45-mm filter before the enzyme-linked immunosorbent assay. Protein samples (100 µL, 10 µg/mL) of NGF (Elabscience, Houston, TX, USA), serotonin (Abcam, Cambridge, UK), CGRP (Elabscience), substance P (R&D SYSTEMS, Minneapolis, MN, USA), NPY (Elabscience), and VIP (Elabscience) were absorbed onto each conjugate-coated plate. Levels of neuromediators in a given sample were measured by comparing its absorption with the standard curve. The minimum detectable concentrations of NGF, serotonin, CGRP, substance P, NPY, and VIP were 15.63 pg/mL, 0.5 ng/mL, 15.63 pg/mL, 78.1 pg/mL, 31.25 pg/mL, and 7.81 pg/mL, respectively.
A specific enzyme-linked immunosorbent assay was performed on extracted proteins to quantify the concentrations of neuromediators in the tear samples of subjects following manufacturer instructions. Tear samples were centrifuged at 3000 g for 10 min and filtered through a 0.45-mm filter before the enzyme-linked immunosorbent assay. Protein samples (100 µL, 10 µg/mL) of NGF (Elabscience, Houston, TX, USA), serotonin (Abcam, Cambridge, UK), CGRP (Elabscience), substance P (R&D SYSTEMS, Minneapolis, MN, USA), NPY (Elabscience), and VIP (Elabscience) were absorbed onto each conjugate-coated plate. Levels of neuromediators in a given sample were measured by comparing its absorption with the standard curve. The minimum detectable concentrations of NGF, serotonin, CGRP, substance P, NPY, and VIP were 15.63 pg/mL, 0.5 ng/mL, 15.63 pg/mL, 78.1 pg/mL, 31.25 pg/mL, and 7.81 pg/mL, respectively.
4
Cytokine and Growth Factor Profiling
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Concentrations of cytokines (IL-2, IL-4, IL-6, IL-10, IL-12(p70), IL-17, IFN-γ, and TNF-α) and growth factors (FGF and VEGF) were measured by using Bio-Plex ProTM cytokine, chemokine, and growth factor assay (Bio-Rad, Hercules, CA). Plasma concentrations of thymic stromal lymphopoietin (TSLP), interferon γ-induced protein 10 (IP-10) (Biolegend, San Diego, CA), platelet factor 4 (PF4) (Sigma-Aldrich, St. Louis, MO), and serotonin (Abcam, Cambridge, UK) were measured by ELISA in accordance with the manufacturer’s instructions.
5
Measuring Neurotransmitters and Hormones in Mice
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Serotonin (Abcam) and 5-HIAA (BioVision) concentration in the mouse brain tissues were measured using enzyme-linked immunosorbent assays, following the manufacturer’s instructions. Total protein level was quantified using the Bradford protein assay (Bio-Rad). Corticosterone concentration in the mouse serum samples was determined using a corticosterone enzyme-linked immunosorbent assay, following the manufacturer’s instructions (Enzo Life Sciences). Blood samples were collected during the dark phase (around 6 to 8 PM).
6
Immunocytochemistry of Differentiated Cells
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Differentiated cells were fixed with 4% paraformaldehyde (PFA) for 20 min at 4°C. Fixed cells were washed three times with Tris buffered saline (TBS). Non-specific binding was blocked and the cells were permeabilized with 0.02% Triton X-100 and 5% normal goat serum (NGS) (PAA, The Cell Culture Company, Somerset, UK), for 1 h at room temperature (RT). Primary antibodies diluted in 1% NGS blocking buffer were added to the cultured cells overnight at 4°C (Oct4, 1:100, Santa Cruz; β-III-tubulin 1:500, Covance; TH, 1:1000, Chemicon; Serotonin, 1:500, Abcam). Negative primary controls consisted of cells treated with blocking buffer without the addition of primary antibodies. On the next day, three TBS washes were applied to the cells followed by incubation with Alexa Fluor secondary antibodies, (Cheshire Sciences, UK) diluted to 1:300 in 1% NGS blocking buffer, for 2 h at RT. Cultures were washed three times with TBS. Coverslips were mounted onto microscope slides using Vectashield hardset mounting medium containing 4', 6-diamidino-2-phenylindole (DAPI) (Vector Labs, Peterborough, UK) to counterstain cell nuclei, before visualisation the following day.
7
Serum Biomarker Quantification by ELISA
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Patient serum was analyzed by ELISA with commercial kits for histamine (Cat. RE59221, IBL International, Germany), human carboxypeptidase A3 (Cat. OKCD01671, Aviva Systems Biology, USA), serotonin (Cat. ab133053, Abcam, UK) and heparin (Cat. abx 258893, Abbexa, USA), according to the manufacturer's instructions.
Nitric oxide was evaluated by a colorimetric method that measures the levels of the breakdown product NO2 -(Griess Reagent System, Cat. G2930, Promega, USA).
Nitric oxide was evaluated by a colorimetric method that measures the levels of the breakdown product NO2 -(Griess Reagent System, Cat. G2930, Promega, USA).
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