cell type: immature DCs (iDCs) treatment: control time: 0h
Treatment protocol
To induce DC maturation, on day 4, 100 ng/mL LPS (Sigma) and 1000 IU/mL IFN-g (R&D Systems) were added. The DCs were harvested at 0, 4, 8 and 24 hours (h) after the addition of LPS and IFN-g. To remove the adherent DCs, 2 mM EDTA-PBS was added to each flask on ice. The harvested cells were pelleted, washed twice with HBSS, and resuspended in RPMI 1640. The total number of cells harvested and their viability was measured microscopically after adding Trypan Blue.
Growth protocol
The elutriated monocytes from each donor were suspended at 6.7 × 10^6/mL with RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (FSC) (Invitrogen), 2 mM L-glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 1% pyruvate (Invitrogen), 100 units/mL penicillin/streptomycin (Invitrogen), and 50 μM 2-mercaptoethanol (Sigma, St Louis, MO). A total of 10 mL of monocyte suspension was cultured in T25 culture flasks (Nalge Nunc International, Rochester, NY) overnight in a humidified incubator with 5% CO2 at 37°C. On Day 1, 2000 IU/mL human IL-4 (R&D Systems) and 2000 IU/mL GM-CSF (R&D Systems) were added to the culture. On Day 3, an additional 2000 IU/mL IL-4 and GM-CSF were added.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the DCs using Trizol (Invitrogen, Carlsbad, CA). RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany).
Label
Cy5
Label protocol
Total RNA (3 μg) from the DCs was amplified into anti-sense RNA (aRNA). While total RNA from PBMCs pooled from the 6 normal donors was extracted and amplified into aRNA to serve as the reference. Pooled reference and test aRNA were isolated and amplified using identical conditions and the same amplification/hybridization procedures to avoid possible interexperimental biases. Both reference and test aRNA were directly labeled using ULS aRNA Fluorescent Labeling kit (Kreatech, Amsterdam, Netherlands) with Cy3 for reference and Cy5 for test samples.
To induce DC maturation, on day 4, 100 ng/mL LPS (Sigma) and 1000 IU/mL IFN-g (R&D Systems) were added. The DCs were harvested at 0, 4, 8 and 24 hours (h) after the addition of LPS and IFN-g. To remove the adherent DCs, 2 mM EDTA-PBS was added to each flask on ice. The harvested cells were pelleted, washed twice with HBSS, and resuspended in RPMI 1640. The total number of cells harvested and their viability was measured microscopically after adding Trypan Blue.
Growth protocol
The elutriated monocytes from each donor were suspended at 6.7 × 10^6/mL with RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (FSC) (Invitrogen), 2 mM L-glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 1% pyruvate (Invitrogen), 100 units/mL penicillin/streptomycin (Invitrogen), and 50 μM 2-mercaptoethanol (Sigma, St Louis, MO). A total of 10 mL of monocyte suspension was cultured in T25 culture flasks (Nalge Nunc International, Rochester, NY) overnight in a humidified incubator with 5% CO2 at 37°C. On Day 1, 2000 IU/mL human IL-4 (R&D Systems) and 2000 IU/mL GM-CSF (R&D Systems) were added to the culture. On Day 3, an additional 2000 IU/mL IL-4 and GM-CSF were added.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the DCs using Trizol (Invitrogen, Carlsbad, CA). RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany).
Label
Cy3
Label protocol
Total RNA (3 μg) from the DCs was amplified into anti-sense RNA (aRNA). While total RNA from PBMCs pooled from the 6 normal donors was extracted and amplified into aRNA to serve as the reference. Pooled reference and test aRNA were isolated and amplified using identical conditions and the same amplification/hybridization procedures to avoid possible interexperimental biases. Both reference and test aRNA were directly labeled using ULS aRNA Fluorescent Labeling kit (Kreatech, Amsterdam, Netherlands) with Cy3 for reference and Cy5 for test samples.
Hybridization protocol
Hybridization was carried out in a water bath at 42°C for 18 to 24 hours and the arrays were then washed
Scan protocol
scanned on a GenePix scanner Pro 4.0 (Axon, Sunnyvale, CA) with a variable photomultiplier tube to obtain optimized signal intensities with minimum (<1% spots) intensity saturation.
Description
DC cell culture in different time point
Data processing
The raw data set was filtered according to a standard procedure to exclude spots below a minimum intensity that arbitrarily was set to an intensity parameter of 200 for both channels
