Fit or senescent MSCs (± IFNγ-activated) were lysed and total RNA was extracted using an RNeasy plus mini kit (QIAGEN). Normalized RNA was used to convert cDNA using Quantitect reverse transcription kit (QIAGEN). Sybr green (Perfecta Sybr green fast mix, Quanta Biosciences) real-time PCR was performed with IDO primer pairs as described previously.22 (link) IDO protein were detected using primary rabbit anti-human IDO1 (1:1,000; EMD Millipore Corporation, Billerica, MA) or rabbit anti-human β-actin (1:1000; Cell Signaling Technology, Inc, Danvers, MA), and secondary horseradish peroxide–coupled goat anti-rabbit IgG h+l (1:10 000; Bethyl Laboratories, Inc, Montgomery, TX). ECL detection system (Amersham Pharmacia Biotech, Piscataway, NJ) was used to detect immunoreactive blots. IDO activity was blocked using 1-methyl-DL-tryptophan (1 mM concentration) (Sigma-Aldrich) in MSC and T-cell coculture.
Secondary horseradish peroxide coupled goat anti rabbit igg h l
Manufactured by Fortis Life Sciences
The Secondary horseradish peroxide–coupled goat anti-rabbit IgG h+l is a detection reagent used in immunoassays. It consists of goat-derived antibodies specific to the heavy and light chains of rabbit immunoglobulin G (IgG), conjugated with the enzyme horseradish peroxidase. This reagent can be used to detect the presence of rabbit IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Ecl detection system, by Cytiva (2 mentions)
Primary rabbit anti human ido1, by Merck Group (2 mentions)
1 methyl dl tryptophan, by Merck Group (2 mentions)
Rabbit anti human β actin, by Cell Signaling Technology (1 mentions)
Perfecta sybr green fastmix, by Quanta Biosciences (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
1 methyl d tryptophan, by Merck Group (1 mentions)
1 methyl l tryptophan, by Merck Group (1 mentions)
Recombinant human ifn γ, by Thermo Fisher Scientific (1 mentions)
2 protocols using secondary horseradish peroxide coupled goat anti rabbit igg h l
1
Quantifying IDO Expression and Activity
2
Quantification of IDO1 and AhR in MSCs
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Approximately 1 million MSCs were harvested from a single 75-cm2 flask at 80% confluency. Cells had been treated for 12h with 50 ng/ml recombinant human IFN-γ (Invitrogen, Carlsbad, CA), and/or 1-methyl-DL-tryptophan, 1-methyl-D-tryptophan, or 1-methyl-L-tryptophan (Sigma-Aldrich, St. Louis, MO). Whole-cell protein lysates were run in a 4-20% polyacrylamide gel electrophoresis apparatus and then transferred to PVDF membrane, which was blocked in 5% non-fat milk in Tris-buffered saline + 0.05% Tween-20. Protein was detected using primary rabbit anti-human IDO1 (1:1000; EMD Millipore Corporation, Billerica, MA), primary mouse anti-human AHR (1:1000; ThermoFisher, Waltham, MA) or primary rabbit anti-human β-actin (1:1000; Cell Signaling Technology, Danvers, MA), and secondary horseradish peroxide-coupled goat anti-rabbit IgG h + l (1:10,000; Bethyl Laboratories, Montgomery, TX). ECL detection system (Amersham Pharmacia Biotech, Piscataway, NJ) was used to detect immunoreactive blots.
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