All mice were euthanized using pentobarbital anesthesia on days zero, seven, 14, and 21. Immediately following euthanization, a 1 ml blood sample was collected from each mouse by cardiac puncture. We measured the plasma acetylcholine concentrations using an Acetylcholine Assay Kit (Cell Biolabs, San Diego, CA, USA). We measured plasma tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) using the respective enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN, USA and eBioscience Inc., San Diego, CA, USA, respectively) according to the manufacturer's instructions. Plasma NO2−/NO3− levels were also measured as a surrogate marker for inducible nitric oxide synthase (iNOS) expression using the NO2/NO3 Assay Kit-C II (Colorimetric) and Griess Reagent Kit (DOJINDO Molecular Technologies, Inc., Kumamoto, Japan) for each nitric oxide (NO) radical.
Acetylcholine assay kit
Manufactured by Cell Biolabs
Sourced in United States
The Acetylcholine Assay Kit is a laboratory tool designed to quantify the levels of the neurotransmitter acetylcholine in biological samples. The kit provides the necessary reagents and protocols to perform colorimetric or fluorometric-based detection of acetylcholine concentrations.
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4 protocols using acetylcholine assay kit
1
Plasma Biomarkers in Mouse Model
2
Acetylcholine Quantification in Liver Tissue
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Liver tissues were collected from ChAT-IRES-Cre mice injected with retroAAV-FLEX-Jaws and ChAT-IRES-Cre mice following 1 h of light illumination. Fifty-five milligrams of liver tissue were homogenized in chloroform/methanol (2:1, v/v). After centrifugation at 4 °C, the homogenate was incubated at room temperature for 1 h on an orbital shaker and phase separation was induced by adding distilled water. The lower organic phase was collected. The upper phase of the sample was re-extracted with chloroform/methanol/water (86:14:1, v/v/v). Organic phases were combined and dried in a vacuum centrifuge. Then, samples were dissolved in chloroform/methanol/water (60:30:4.5, v/v/v). Acetylcholine levels were quantified using the Acetylcholine assay kit (Cell Biolabs, Inc., STA-603).
3
Serum Neurotransmitter Quantification
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A laparotomy was performed under isoflurane anesthesia to collect blood from the abdominal aorta. Collected blood was allowed to clot overnight at room temperature and then centrifuged at 2,000 g for 15 min to separate the serum.
Serum 5‐HT levels were determined using a Serotonin Research ELISA Kit (BA E-5900; ImmuSmol SAS, Pessac, France), and serum DA levels were determined using a Dopamine Research ELISA Kit (BS E-5300; ImmuSmol SAS, Pessac, France). Both neurotransmitters were extracted from serum samples, acylated, and assayed according to the manufacturer’s instructions.
Ach was assayed in filtered serum samples using an Acetylcholine Assay Kit (STA-603; Cell Biolabs, Inc. USA) according to the manufacturer’s instructions.
Serum 5‐HT levels were determined using a Serotonin Research ELISA Kit (BA E-5900; ImmuSmol SAS, Pessac, France), and serum DA levels were determined using a Dopamine Research ELISA Kit (BS E-5300; ImmuSmol SAS, Pessac, France). Both neurotransmitters were extracted from serum samples, acylated, and assayed according to the manufacturer’s instructions.
Ach was assayed in filtered serum samples using an Acetylcholine Assay Kit (STA-603; Cell Biolabs, Inc. USA) according to the manufacturer’s instructions.
4
Quantification of Acetylcholine Levels
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The concentration of ACh was quantified using an Acetylcholine Assay Kit (Cell Biolabs Inc., San Diego, CA, USA) in accordance with the manufacturer’s protocols. Briefly, the mid colon from mice (n = 5) in subset group was homogenized in ice-cold 1× PBS (pH 7.2–7.4) using a glass homogenizer (Sigma-Aldrich Co.); the homogenate was stored at −70 °C prior to ACh analysis. For analysis, the homogenate sample (or standards) and the ACh reaction mixture were incubated on an orbital rotator for 45 min at RT in a 96-well plate protected from light. Color alteration within the plate wells was determined using a Vmax plate reader (Molecular Devices) at 570 nm (excitation) and 600 nm (emission).
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