The amount of L-kynurenine in culture supernatants was measured with a spectrophotometric analysis, as previously described.23 (link) Briefly, Mo-DC were washed, resuspended in Hanks buffered saline solution supplemented with 500 µM L-tryptophan (Sigma-Aldrich) and incubated (where indicated) with or without the IDO inhibitors 1-MT-D or –L (1 mM, Sigma-Aldrich). Supernatants were harvested after 4 h and mixed with 30% trichloroacetic acid (2:1), vortexed and centrifugated at 8000 g for 5 min. Subsequently, this solution was added to Ehrlich reagent (1:1, Sigma-Aldrich) in a 96-well plate. Triplicate samples were run against a standard curve of defined kynurenine concentrations (0–100 µM; Sigma-Aldrich). Optical density was measured at 490 nm, using a Multiskan EX microplate reader (Thermo Electron Corporation, Vantaa, Finland).
Ehrlich reagent
Manufactured by Merck Group
Sourced in United States
Ehrlich reagent is a laboratory chemical used for various analytical applications. It is a colorimetric test reagent employed to detect the presence of certain organic compounds, such as indoles and some heterocyclic compounds. The reagent is prepared by dissolving p-dimethylaminobenzaldehyde in a mixture of hydrochloric acid and ethanol.
Automatically generated - may contain errors
Lab products found in correlation
Trichloroacetic acid, by Merck Group (6 mentions)
L kynurenine, by Merck Group (4 mentions)
L tryptophan, by Merck Group (3 mentions)
N acetylglucosamine, by Merck Group (2 mentions)
P dimethylbenzaldehyde, by Merck Group (2 mentions)
Kynurenine, by Merck Group (2 mentions)
Microplate reader, by Molecular Devices (2 mentions)
Masson s trichrome staining kit, by Merck Group (2 mentions)
Chloramine t, by Merck Group (2 mentions)
Kynurenine standard, by Merck Group (2 mentions)
Glacial acetic acid, by Merck Group (2 mentions)
1 d mt, by Merck Group (1 mentions)
Multiskan ex microplate reader, by Thermo Fisher Scientific (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Hydroxyproline, by Merck Group (1 mentions)
Tgf β1, by BioLegend (1 mentions)
Anti p smad2 3, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Merck Group (1 mentions)
Anti smad2 3, by Cell Signaling Technology (1 mentions)
Anti fibronectin, by Santa Cruz Biotechnology (1 mentions)
18 protocols using ehrlich reagent
1
Spectrophotometric Analysis of L-kynurenine in Monocyte-Derived Dendritic Cells
2
Antimicrobial Activity Screening of Natural Compounds
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Cinnabarinic acid (CA, ≥98%, CAS: 606–59-7), Ehrlich reagent (CAS: 100–10-7), and N-acetylglucosamine (CAS: 7512-17-6) were purchased from Sigma-Aldrich (Shanghai, China). Sugarcane bagasse, wheat straw, rice straw, water hyacinth, and wood chip were purchased from a local market and dried to constant weight at 60°C before use. Other chemical reagents, unless otherwise specified, were all analytical grade.
Indicator strains, including Bacillus subtilis SYBC-hb1, Bacillus subtilis SYBC-hb5, Bacillus licheniformis SYBC-hb2, Bacillus pumilus SYBC-hb4, Bacillus thuringiensis SYBC-hb7, Bacillus amyloliquefaciens Y1-A3, Bacillus megaterium H021-A1, Bacillus cereus SYBC-hb8, Lysinibacillus sp. H021-S8, Staphylococcus kloosii H008-B4, Staphylococcus aureus, Escherichia coli J159, Serratia sp. L1, Serratia sp. L2, and Serratia sp. L3, were obtained from Biocatalysis and Transformation Biology lab at Jiangnan University (Wuxi, China).
Indicator strains, including Bacillus subtilis SYBC-hb1, Bacillus subtilis SYBC-hb5, Bacillus licheniformis SYBC-hb2, Bacillus pumilus SYBC-hb4, Bacillus thuringiensis SYBC-hb7, Bacillus amyloliquefaciens Y1-A3, Bacillus megaterium H021-A1, Bacillus cereus SYBC-hb8, Lysinibacillus sp. H021-S8, Staphylococcus kloosii H008-B4, Staphylococcus aureus, Escherichia coli J159, Serratia sp. L1, Serratia sp. L2, and Serratia sp. L3, were obtained from Biocatalysis and Transformation Biology lab at Jiangnan University (Wuxi, China).
3
Quantifying Kynurenine Levels for IDO Activity
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Since kynurenine is the product of IDO-dependent catabolism of tryptophan, the biological activity of IDO was evaluated by monitoring the level of kynurenine in C-MSC culture. One hundred microliters of culture supernatant was mixed with 50 μL of 30% trichloratic acid (Sigma-Aldrich), vortexed, and centrifuged at 10,000 × g for 5 min. Then, 75 μL of the supernatant was added to an equal volume of Ehrlich reagent (100 mg p–dimethylbenzaldehyde (Sigma-Aldrich) in 5 mL glacial acetic acid) in a 96-well plate and incubated at room temperature for 10 min. The absorbance at 492 nm was determined. The concentration of kynurenine was quantified using a standard curve generated from defined kynurenine (Sigma-Aldrich) concentrations (0–150 μM).
4
Quantifying Renal Hydroxyproline Content
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For the measurement of renal hydroxyproline content, a previously described method was used32 (link). In brief, snap-frozen kidney specimens (200 mg) were weighed, hydrolyzed in HCl (6 M; Merck, USA) for 12 h at 100 °C. Next, they were oxidized with Chloramine-T (SRL, India). Next, Ehrlich reagent (Sigma, USA) was added which resulted in the formation of a chromophore. Absorbance was measured at 550 nm. Data were normalized to kidney wet weight.
5
IDO Activity Measurement in hMSCs
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The biological activity of IDO was calculated by measuring the level of kynurenine in supernatant collected from different preconditioned tissue-specific hMSCs. Briefly, 100 μl of collected cell culture supernatant were added to the Eppendorf tube and 50 μl of 30% trichloroacetic acid (Sigma, USA) were added, the tube was vortexed and centrifuged at 8000 g for 5 min. Then, 75 μl of the supernatant was transferred with an equal volume of Ehrlich reagent (100 mg p-dimethyl benzaldehyde in 5 ml glacial acetic acid) (Sigma, USA) to a 96-well microtiter plate and recorded the absorbance at 490 nm [26 (link)]. Experiment was performed at least in three independent setups.
6
Measurement of IDO Activity in E-SF-MSCs
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IDO activity was measured as previously described [15 ]. Cultures were harvested and 2.5 × 104 E-SF-MSCs with or without induction of an RA-like inflammatory milieu were cultured for 4 days. Then, the cells were supplemented with 100 μM L-tryptophan (Sigma) for 4 h. The supernatant was harvested and mixed with 30% trichloroacetic acid (Sigma) before an additional incubation at 50°C for 30 min. This solution was diluted in Ehrlich reagent (1:1) (Sigma, USA), and the optical density was measured at 492 nm using a microplate reader (Molecular Devices). Serially-diluted l -kynurenine (Sigma) made with fresh culture medium was used as the standard.
7
Withaferin A Attenuates Pulmonary Fibrosis
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Withaferin A was procured from Aptus laboratories (Hyderabad, India) and TGF-β1 was obtained from Bio-legend (United States); Bleomycin sulfate was procured from Cipla labs (India); Masson’s trichrome staining kit, Sirius red, Chloramine-T, Hydroxyproline, and Ehrlich reagent were procured from Sigma-Aldrich, Anti-ZO-1, anti-E-cadherin, anti-Smad 2/3, anti-p Smad 2/3, anti-vimentin, anti-NF-κB p65, anti-p VEGF, anti-p p38 MAPK, anti-p FAK, and anti-p PLCγ1 were procured from Cell Signaling Technology, while anti-Col 1A2, anti-Col 3A1, anti-smooth muscle actin, anti-CTGF, anti-fibronectin, and anti-TGF-β1 were obtained from Santa Cruz Biotechnology (United States). ELISA kits were purchased from eBioscience, United States. TGF-β bioplex kit was procured from Merck-Millipore. Rest of the chemicals and reagents were of analytical grade and obtained from commercially available sources.
8
Kynurenine Production Assay for IDO Activity
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IDO activity, represented as concentration of kynurenine produced, was analyzed by a spectrophotometric assay reported earlier31 (link). Cell-free spent culture media were harvested from primary macrophage or macrophage/MSC co-cultures and used immediately for assay. To 120 μl of spent medium, 60 μl of 30% trichloroacetic acid [Sigma Aldrich] was added, mixed thoroughly and centrifuged at 8000 g for 5 min at room temperature. From the clarified supernatant, 85 μl was transferred immediately to 96-well plates and 85 μl of 1% Ehrlich reagent prepared in glacial acetic acid [Sigma Aldrich] was added and incubated for 10 min at room temperature. Absorbance was read at 490 nm on a multimode plate reader [EnsightTM, Perkin Elmer]. Concentrations were determined against a kynurenine standard [Sigma].
9
Fibrosis Biomarker Evaluation Protocol
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BLM sulfate (Cipla Labs), chloramine-T, trans-L-Hydroxyproline, Masson's trichrome staining kit, Griess reagent, Ehrlich reagent, reduced glutathione (GSH), 5,5-dithio-bis (2-nitrobenzoic acid), 2-thiobarbituric acid (TBA), bovine serum albumin (BSA), sodium dodecyl sulfate (SDS), glacial acetic acid, sodium nitrite, and Bradford reagents were obtained from Sigma-Aldrich, USA. All other chemicals were of analytical grade and were obtained commercially. Anti-α-smooth muscle actin (SMA), anti-E-cadherin, horseradish peroxidase (HRP) anti-mouse and anti-rabbit antibodies were obtained from Santa-Cruz Biotechnology Inc. Anti-vimentin and anti-fibronectin primary antibodies were procured from Cell Signaling Technology.
10
Utilization of Chemical Reagents in Research
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The curcumin, kynurenine standard, gallic acid, tannic acid, and Ehrlich reagent were products of Sigma Chemicals Co. (St. Louis, Missouri, USA). The agar and broth came from HiMedia (Mumbai, India). The chemical reagents were of analytical grade and were used as supplied. Every compound used in this work was dissolved using Dimethyl sulfoxide (DMSO).
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