Project description:Macrophage polarization factors to direct digit number and identity in the treated macrophages. We have characterized the macrophage-dependent regulatory genes network through a combination of whole genome RNA sequencing and transcriptional profiling in the phorbol and non-thermal plasma treated THP-1 monocytes. In this dataset, we include the expression data obtained from differentiated macrophages (THP-1) using a very lose doses of non-thermal plasma, as well as THP-1 which were treated only with phorbol (PMA). These data are used to obtain genes that are differentially expressed in response to macrophage polarization by plasma treatments.
Project description:Human MSCs were cocultured for 18 hours with macrophages which had been differentiated from human peripheral blood-derived monocytes by PMA and sequentially stimulated by 2 μg/mL LPS (InvivoGen) for 3 hours and 5 mM ATP (InvivoGen) for 45 minutes. Normal MSCs without macrophage coculture and MSCs cocultured with activated macrophages were assayed by miRNA arrays.
Project description:Macrophages are sentinels of the immune system and THP1 monocytic cells are one of the widely used models to study immune responses in macrophages. Several monocyte-to-macrophage differentiation protocols exist with phorbol 12-myristate-13-acetate (PMA) being widely used and accepted. However, the concentrations and durations of PMA treatment to induce differentiation varies widely in published literature. In this study, we determined the proteome expression dynamics of THP1 macrophages differentiated from monocytes by three commonly used PMA-based differentiation protocols using dimethyl labeling-based quantitative proteomics analysis. Our analysis shows that variations in PMA concentration and varying periods of rest post-stimulation result in downstream differences in proteome expression and cellular processes. We demonstrate that these differences result in altered expression of cytokines upon stimulation with different TLR ligands. Together, these findings provide a valuable resource that significantly expands the knowledge of protein expression dynamics in in vitro models of macrophages which in turn has profound impact on the immune responses being studied.
Project description:THP-1 monocytes were differentiated, via PMA treatment, to macrophages. THP-1 macrophages were then treated with a mixed-oxysterol treatment (7-ketocholesterol and 7beta-hydroxycholesterol) for 24 hours. The mixed-oxysterol treatment was mixed to a ratio that represents the cytotoxic oxysterol content of human atherosclerotic plaque. Control and treated macrophages were analysed using both 2-DE with peptide mass-fingerprinting and tandem-mass spectrometry.
Project description:The data is supplementary to the RNA-seq analysis of LPS and palmitate stimulation of THP-1 macrophages (E-MTAB-6064), where palmitate for the cell treatment was dissolved in sodium hydroxide and coupled with BSA at a molar ratio 7.5:1. Here we stimulated THP-1 macrophages with the corresponding concentration of BSA (4%) and NaOH (0.4 mM) in the presence of 10 nM phorbol 12-myristate 13-acetate (PMA) for 24 hours added to the standard RPMI 1640 medium with 10% fetal bovine serum (FBS) to estimate the effects and compared them with unstimulated THP-1 macrophages cultured in RPMI 1640 medium with 10% FBS and 10nM PMA.
Project description:THP-1 cells were differentiated to macrophages with the phorbol 12-myristate 13-acetate (PMA) for a total time of 28 hours. To understand the effects of Angiotensin II (AngII) and of AngII in the presence of the thiazolidinedione pioglitazone (Pio), after 4 hours of PMA-induced differentiation of THP-1 cells (when cells became adherent), the PMA-containing medium was supplemented with AngII (1mg/ml), AngII (1 mg/ml) with Pio (1 uM), or no supplement for another 24 hours. At this time point the cells were harvested, RNA was extracted and used for a microarray-based gene profiling analysis of THP-1 cells, under the three conditions: PMA, AngII and AngII with Pio.
Project description:Human monocyte THP-1 cells obtained from ATCC were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) containing 10% FBS and supplemented with 10 mM Hepes (Gibco BRL). THP-1 was differentiated into macrophages by 24-h incubation with 160 nM phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO) followed by 24-h incubation in RPMI medium. Macrophages were further polarized to M1 macrophages by incubation with 10 pg/ml of lipopolysaccharide (LPS; Sigma) and 20 ng/ml of interferon (IFN)-γ (R&D Systems, MN) and are referred to as M(LPS+IFN-γ) cells. M2 macrophages were obtained by incubation with 20 ng/ml of interleukin (IL)-4 (R&D Systems) and are referred to as M(IL4) cells. To test the represented polarization marker of PMA differentiated-THP-1 macrophages stimulated with 20 ng ml(-1) IFNγ + 10 pg ml(-1) LPS and 20 ng ml(-1) IL-4, which are known to influence macrophage polarization in vetro into the M1 and M2 state, respectively. We used microarrays to detail the gene expression pools to identify distinct M1 and M2 state during this process.
Project description:As part of our study in understanding the role of SP140 in inflammatory pathways in macrophages, we inhibited SP140 mRNA using siRNA. Peripheral blood mononuclear cells (PBMCs) were obtained from whole blood of healthy donors (from Sanquin Institute Amsterdam or from GSK Stevenage Blood Donation Unit) by Ficoll density gradient (Invitrogen). CD14+ monocytes were positively selected from PBMCs using CD14 Microbeads according to the manufacturer’s instructions (Miltenyi Biotec). CD14+ cells were differentiated with 20 ng/mL of macrophage colony-stimulating factor (M-CSF) (R&D systems) for 3 days followed by 3 days of polarization into classically activated (inflammatory) M1 macrophages (100 ng/mL IFN-γ; R&D systems). M1 macrophages were transfected with siGENOME human smartpool SP140 siRNA or non-targeting scrambled siRNA for 48h with DharmaFECT™ transfection reagents according to manufacturer’s protocol (Dharmacon). The cells were left unstimulated or stimulated with 100 ng/mL LPS (E. coli 0111:B4; Sigma) for 4h (for qPCR) or 24h (for Elisa). The cells were lysed (ISOLATE II RNA Lysis Buffer RLY-Bioline) for RNA extraction.150 ng total RNA was labelled using the cRNA labelling kit for Illumina BeadArrays (Ambion) and hybridized with Ref8v3 BeadArrays (Illumina). Arrays were scanned on a BeadArray 500GX scanner and data were normalized using quantile normalization with background subtraction (GenomeStudio software; Illumina). This submission only contains processed data