To measure IDO-1 activity in mammalian tissues, we used the IDO1 Activity Assay Kit (Cat# Ab235936; Abcam; Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, we harvested complete female reproductive tracts, and the entire zone containing the endocervix and ectocervix was isolated, macerated with liquid nitrogen, and homogenized in 500 μL IDO-1 ice-cold assay buffer, followed by centrifugation (10,000× g, 15 min, 4 °C), and then the supernatant was collected. After that, 50 μL reaction premix (2×) was prepared, mixed with 15 μL of the test sample, and made up to 90 μL with IDO1 assay buffer. Next, 10 μL of the 1 mM IDO-1 substrate solution was added to each assay well, and the plate was incubated at 37 °C in a dark environment for 45 min. Then 50 μL of the fluorogenic developer solution was added, the plate was incubated at 45 °C for 3 h, and the fluorescence (Ex/Em = 402/488 nm) was measured.
Ido1 activity assay kit
Manufactured by Abcam
Sourced in United States
The IDO1 Activity Assay Kit is a tool used to measure the enzymatic activity of indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme involved in the catabolism of the amino acid tryptophan. The kit provides a quantitative colorimetric method for determining IDO1 activity in biological samples such as cell lysates or tissue homogenates.
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5 protocols using ido1 activity assay kit
1
Quantifying IDO-1 Activity in Mammalian Tissues
2
Quantifying Prostaglandin E2 and IDO Activity
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Prostaglandin E2 (PGE2) production was detected by single Elisa assay (Cayman Chemical). Moreover, the enzymatic activity of indolamine 2,3-dioxygenase (IDO), a cytoplasmic hemoprotein that oxidizes tryptophan yielding N-formylkynurenine (NFK), was evaluated on cell lysates by an IDO1 Activity Assay Kit (Abcam) according to manufacturer’s instruction. IDO metabolic activity was obtained by interpolating the fluorescence values obtained to the standard curve corresponding to the NFK concentration. The metabolic activity was then obtained as pmole of L-tryptophan metabolized by IDO during the reaction time. The level of expression of the indolamine 2,3-dioxygenase gene (primer sequence are reported in supplementary table 1 ) was evaluated by real-time PCR, as previously described.
3
IDO1 Activity Measurement Protocol
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IDO1 activity was measured with IDO1 Activity Assay Kit (Abcam, ab235936) according to the operation manual.
4
Quantifying Brain IDO Activity
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To determine the IDO activity of mouse brain, the concentration of kynurenine and tryptophan was measured by high performance liquid chromatography (HPLC) analysis. At 24 hours post injection, mouse brain homogenates were prepared in the lysis buffer containing 25 mM Tris-Cl (pH 7.5), 1% NP-40, 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitors. Homogenates were normalized by Bradford assay and used to quantify the level of kynurenine and tryptophan by HPLC-MS/MS analysis. Liquid-liquid extraction using water-saturated ethyl acetate was performed, and aqueous layer was injected into HPLC-Tandem mass spectrometry (MS/MS) on phenomenex Kinetex C18 column to separate kynurenine and tryptophan in selective reaction monitoring mode (kynurenine m/z 209 > 192; tryptophan m/z 205 > 118). To further assess the enzyme activity of IDO in the hippocampal homogenates of mouse brain, IDO1 activity assay kit (BioVision) was used according to the manufacturer’s instruction. One unit of IDO1 activity is the amount of enzyme that generates 1 μmol of detected N-formylkynurenine per minute by oxidation of 1 μmol L-tryptophan at 37°C.
5
Quantification of IDO1, Trp, and Kyn in Cell Culture
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The concentration of IDO1 from culture medium was detected by ELISA (Cat# JL20020, JiangLai, China) according to the manufacturer’s protocol. The concentration of Trp (Cat# BWB51529, National Institutes for Food and Drug Control) and Kyn (Cat# K8625, Sigma) from culture medium were measured by using high performance liquid chromatography (HPLC). The samples were separated on a Welch Ultimate AQ-C18 column (250 mm × 4.6 mm, 5 μm) with a mobile phase of acetonitrile and 15 mM potassium phosphate buffer (8:92, v/v) at a flow rate of 1.0 mL/min with a column temperature 35 °C. Potassium phosphate buffer was adjusted to pH 3.6 with acetic acid. The UV detection wavelengths for Trp and Kyn are 280 nm and 360 nm, respectively. Thirty microliters of HT-29 and HCT-116 cell lysate was used to assess IDO1 function according to the manufacturer’s protocol (IDO1 Activity Assay Kit, Cat# K972, Biovision).
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