cell type: blood monocytes treatment: gm-csf (100 ng/ml) for 5 days gender: male.
Treatment protocol
Monocytes were differentiated into M1 and M2 macrophages by incubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF)(100 ng/ml, R&D Systems, Minneapolis, MN, USA), respectively, for 5 days in RPMI 1640 culture medium supplemented with glutamine, gentamycine, pyruvate, and 10% fetal calf serum. To induce cellular accumulation of LDL, macrophages were incubated with oxidized LDL (50 μg/ml) for 24 hours. Accumulation of LDL was quantified by Oil-Red-O staining according to routine procedures and by measuring apolipoprotein B (apoB) in cell lysates by ELISA.
Growth protocol
Blood was obtained from healthy volunteers by venipuncture and collected in EDTA Vacutainer tubes. Mononuclear cells were isolated by Ficoll-Paque (Pharmacia Biotech AB, Uppsala, Sweden) density-gradient centrifugation. Monocytes were isolated from mononuclear cells by adherence to plastic in 12-well cell culture clusters (Corning, NY, USA). No restrictions on the diet of the volunteers were imposed.
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared from cells using TRIzol reagent and whereafter purified total RNA was isolated using Qiagen RNEasy columns. RNA integrity was checked on chip analysis (Agilent 2100 bioanalyzer, Agilent Technologies, Amsterdam, the Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0).
Label
biotin
Label protocol
The Affymetrix GeneChip WT Sense Target Labeling and Control Reagents kit (Affymetrix, Santa Clara, CA; P/N 900652) was used for the preparation of labelled cDNA from 100 ng of total RNA without rRNA reduction. A detailed description can be found in the User Manual, Chapter 3 (P/N 701880, revision 5).
Hybridization protocol
Hybridisation of 5.5ug labelled cDNA was done overnight for 17 hours, at 60 rpm, at 45ºC in a Hybridisation Oven 640 (Affymetrix). The protocol was conducted as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5).
Scan protocol
Washing and staining of the arrays was done on a Affymetrix 450 fluidics stations using the protocol FS450_0001, as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5). Arrays where then scanned on an Affymetrix 3000 7G scanner, as described in the Genechip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Arrays: Washing, Staining and Scanning (P/N 701028, revision 5).
Macrophage heterogeneity in human carotid artery atherosclerotic plaques: identification of novel markers for M1 and M2 that are independent of the activation status
Data table header descriptions
ID_REF
VALUE
RMA signal (as log2), from Bioconductor library 'Oligo' (v1.12), with targets = 'core'.
