|
|
Blood, Vol. 108, Issue 1, 218-227, July 1, 2006
Hepatocyte growth factor favors monocyte differentiation into regulatory interleukin (IL)-10++IL-12low/neg accessory cells with dendritic-cell features
Blood Rutella et al.
108: 218
Supplemental materials for: Rutella et al
Supplemental Methods Real-Time PCR Kinetic PCR for each gene of interest was performed on a LightCycler™ (Roche Diagnostics) by using SYBR® Green I as a double-strand DNA-specific binding dye and continuous fluorescence monitoring.26 Amplification was carried out in a total volume of 20 µl containing 0.5 µM of each primer, 10 µl 2× QuantiTect SYBR Green PCR Master Mix (Qiagen, Milan, Italy) and 1 µl of diluted cDNA prepared as above described. The PCR reactions were cycled 55 times after initial denaturation at 95°C for 15 minutes with the following parameters: denaturation (95°C, 15s); annealing (55°C, 25s); and extension (72°C, 15s) with temperature transition rate of 20°C/s. Fluorescence was acquired at the end of annealing step. Melting curve analysis of amplification products was performed at the end of each PCR reaction by cooling the samples to 45°C and then increasing the temperature to 95°C at 0.1°C/s. Fluorescence was acquired every 0.1s. Control reaction for product identification consisted of: (i) analysing the melting peaks; and (ii) determining the length of the PCR products (bp). The latter reaction was performed after completing the PCR reaction on the LightCycler system and centrifuging the content of the glass capillaries in a plastic tube at 200g. Twenty µl of each PCR product were visualized after agarose gel electrophoresis and ethidium bromide staining. To characterize the range of process efficiency linearity and construct the standard curves of crossing-point (Cp) values of the target sequence and reference sequence for the relative quantification, 4× serial dilution of cDNA prepared by 1µg of RNA extracted from CD4+CD25— T cells from 3 healthy controls were tested in triplicate. Each dilution from the standard curve was analysed with the LigthCycler PCR using primer sets for  -actin (reference) and CCL1, CCL2 CXCL1 and INDO (target) cDNA. For each set of primers, a Cp cycle number was determined for each dilution of the standard curve. Linear regression analysis of the logarithm of the dilution factor vs. the crossing point cycle number generated a standard curve for each transcript-specific primer set. From each primer set’s standard curve, a Cp cycle number could be converted into a relative amount of cDNA. PCR efficiency was calculated, from each linear regression, using the equation 101/slope — 1 × 100.
Files in this Data Supplement:
- Table S1. The 672 significantly changing genes (PDF, 124 KB)
- Table S2. Genes up-regulated by HGF treatment (PDF, 123 KB)
- Table S3. Primers used for Real-time kinetic PCR (PDF, 36.1 KB)
- Figure S1. Kinetics of monocyte activation with either GM-CSF+IL-4 or HGF (PDF, 43.5 KB) -
Monocytes and monocyte supernatants were harvested on days 2, 4 and 6 of culture and were used for flow cytometry and ELISA, respectively. Panel A: Expression of informative DC antigens on freshly isolated (day 0) and cytokine-exposed monocytes. One representative experiment out of 3 with similar results is shown. The percentage of positive cells is indicated in each histogram. Panel B: Levels of IL-12p70 in monocyte culture supernatants collected on days 1, 2, 4 and 6 of culture. Data are representative of 3 independent experiments with similar results.
- Figure S2. Expression of informative differentiation/maturation antigens on peripheral monocytes cultured with GM-CSF+IL-4 or with HGF (PDF, 27.5 KB) -
Monocytes were cultured with GM-CSF+IL-4 or with HGF as detailed in “Materials and Methods.” One representative experiment out of 4 with similar results is shown. The Mean Fluorescence Intensity of a given antigen is indicated in each histogram.
- Figure S3. Detection of FoxP3 protein in CD4+CD25+ T cells from normal control subjects (PDF, 25.1 KB) -
Expression of FoxP3 was measured by flow cytometry in freshly isolated CD4+CD25— and CD4+CD25+ T cells, as negative and positive controls, respectively. Data from one representative experiment out of 4 with similar results are shown. The percentage of CD4+CD25++FoxP3+ cells is indicated.
- Figure S4. Real-time PCR experiments on GM4-DCs, HGFMo and cytokine-untreated monocytes (PDF, 12.1 KB) -
Monocytes were cultured either in the presence of GM-CSF+IL-4 (GM4-DCs) or in the presence of HGF (HGFMo), as detailed in Materials and Methods. Control cultures were performed with monocytes cultured in the absence of exogenous cytokines (Mo). Primer sets were designed using the LightCycler Probe Design Software (version 1.0; Roche Diagnostics, Mannheim, Germany) and the sequences available in the GeneBank™ data base. The specific oligonucleotide primer sequences are listed in Table S3. Real-Time PCR studies were performed as detailed in the methods above. The expression of the gene of interest in HGFMo was normalized to 1. Gene expression in cytokine-untreated monocytes and GM4-DCs was depicted as relative to 1.
|
|
