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Blood, Vol. 106, Issue 7, 2375-2381, October 1, 2005
A two-step induction of indoleamine 2,3 dioxygenase (IDO) activity during dendritic-cell maturation
Blood Braun et al.
106: 2375
Supplemental materials for: Braun et al, Vol 106, Issue 7, 2375-2381
Files in this Data Supplement:
- Figure S1. Setup of the qRT-PCR for IDO mRNA (PDF, 53.8 KB) -
Amplification plots showing increase in fluorescence with cycle number. Results are shown for increasing amounts of cDNA prepared from total human PBMC. Fewer cDNA copies resulted in more cycles being required for the generation of a fluorescent signal. Following real-time PCR, amplification products were subjected to melting-curve analysis. The presence of a single peak indicated high specificity of the PCR and excluded parasitic primer dimers as a source of fluorescent signal.
- Figure S2. Correlation between HPLC and colorimetric assays for analysis of kynurenine concentrations (PDF, 83.4 KB) -
(A) HPLC profiles of iDC and mDC (TNF
 + PGE2) culture supernatants are shown. Tryptophan, due to its aromatic cycle, is detected at 280 nm (turquoise line) while both compounds can be measured at 254 nm (red line). Peaks corresponding to the elution time for each compound are indicated with arrows. The thin blue line represents the percentage of elution buffer along time. Dotted lines on the mature DC profile represent baselines used to calculate the area under the peaks. In iDCs, no IDO activity can be detected and the peaks for tryptophan are maximal. mDCs catabolize tryptophan into kynurenine, which is eluted at a lower percentage of elution buffer than tryptophan (determined using standards, data not shown). The area of the peaks at 254 nm are proportional to the quantity of tryptophan and kynurenine present in the sample. The calculation of the tryptophan and kynurenine concentrations in each sample is evaluated using a standard curve (samples containing increasing amounts of kynurenine and decreasing amounts of tryptophan were used, thus maintaining a total concentration of analyte equal to 100 μM, not shown). (B) A standard curve is obtained for each colorimetric assay using kynurenine concentrations between 0 and 100 µM. In a representative experiment, the linear extrapolation of the curve, R2 = 0.9995. Of note, tryptophan does not interfere with the assay (data not shown). (C) Kynurenine levels were measured in parallel by HPLC and the colorimetric assay in 26 different samples. The graph displays the results for 4 samples run from a single donor. Values for the colorimetric assay represent the mean ± SD of two independent wells. (D) Plotted is the correlation for all 26 samples that were analyzed by both methods. A linear correlation curve was obtained (P = 0.755) indicating that there is no significant departure from linearity.
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