PMNs, monocytes, and mDCs were stimulated with LPS (100 ng/ml) for 6 h and CD4+ and CD8+ T cells, B-cells, and NK cells were stimulated with PHA (5 ug/ml) for 24 h. The stimulation condition for pDCs was CpG-ODN 2216 (5 ug/ml) for 16 h as previously reported.
Growth protocol
Leukocytes were then separated into PMNs and mononuclear cells (MNCs) by density gradient centrifugation in Ficoll-Plaqueä (Amersham Pharmacia) at a ratio of 2:1 at 1500 rpm for 30 minutes at 20°C. After centrifugation over a Ficoll cushion, MNCs were washed and counted on a hemocytometer by trypan blue staining. The PMN fraction in MNCs was less than 1% in adult and neonate samples.CD4+ T-cells, CD8+ T-cells, CD14+ monocytes or CD56+ NK cells were separated from MNCs with the IMag Cell Separation Systems (BD Biosciences, San Jose, CA, USA) following the protocol supplied by the manufacturer. In brief, per 2×107 of mononuclear cells pellet was suspended in 100 µl of anti-human CD4, CD8, CD14 or CD56 magnetic particles (BD Biosciences) and incubated at room temperature for 30 min. Then the labeled cells were resuspended in 1X BD IMag™ buffer and the tubes were placed in the BD Imagnet™ (BD Biosciences) for 10 min. With the tube in the BD IMagnet™, the supernatant was removed to discard the undesired leukocytes. To maximize purity, the process was repeated three times. Then the indicated cell fraction was carefully washed and suspended in PBS or medium. For pDCs isolation, dead cells were initially depleted by magnetic negative selection with Dead Cell Removal Kit (Miltenyi. Biotec, Bergisch Gladbach, Germany), then per 5×107 of mononuclear cells pellet was isolated with 50 µl of CD304 (BDCA-4)-microbeads (Miltenyi Biotech) and magnetic columns according to the manufacturer’s instructions. The mDCs were isolated by magnetic positive selection with CD1c (BDCA-1)-microbeads and a magnetic column (Miltenyi Biotech) following the depletion of CD19+ cells. The purity of isolated cells was confirmed by flow cytometry, and all isolated cells demonstrated greater than 90% purity.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the TRIzol® Reagent (Invitrogen, USA) according to the manufacturer’s instructions. Purified RNA was quantified at OD260 nm using a ND-1000 spectrophotometer (Nanodrop Technology, USA) and qualitatively analyzed using a Bioanalyzer 2100 (Agilent Technology, USA) with RNA 6000 nano lab-chip kit (Agilent Technologies, USA).PMNs, CD4+ T cells, CD8+ T cells, CD14+ monocytes, and CD56+ NK cells were isolated from healthy adult peripheral or cord blood obtained from pools of five donors each. The BDCA1+ mDCs were obtained from pools of 10 healthy adult peripheral or cord blood donors. The BDCA4+ pDCs were obtained from pools of 19 healthy adult peripheral blood and 31 cord blood donors for its little amount.
Label
Cy3
Label protocol
Total RNA (0.1 μg) was dephosphorylated and labeled with pCp-Cy3 using the Agilent miRNA Complete Labeling and Hyb Kit (Agilent Technologies, USA, microRNA Spike- In Apply).
Hybridization protocol
Hybridization buffer (2X) (Agilent Technologies, USA) was added to the labeled mixture at a final volume of 45 ml. The mixture was heated for 5 min at 100°C and immediately cooled to 0°C. Each 45 ml sample was hybridized onto an Agilent human miRNA Microarray R19 (Agilent Technologies, USA) at 55°C for 20 h.
Scan protocol
After hybridization, slides were washed for 5 min in Gene Expression Wash Buffer 1 at room temperature, and then washed for 5 min in Gene Expression Wash Buffer 2 at 37°C. Microarrays were scanned using the Agilent microarray scanner (Agilent Technologies, model G2505C) at 535 nm for Cy3.
Description
microRNA after 24 hr in PHA CD8 (isolated from healthy adult peripheral from pools of 5 donors )
Data processing
Feature Extraction (Agilent Technologies) software version 10.7.3.1 was used for image analysis. If both channels produced intensities less than 100 for a given microRNA, that spot was filtered out. For spots with one channel intensity less than 100 and the other 100 or greater, the signal that was less than 100 was set to 100 prior to calculation of the signal ratio. Hierarchical clustering was performed using Genesis software (Graz University of Technology) and Pearson’s correlation was calculated as the distance metric.
