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Expression profile of select inflammation-related genes after miR-466l overexpression or knockdown in human macrophages

ABSTRACT: Human Peripheral blood mononuclear cells (PBMC) were isolated from healthy human whole blood by density gradient centrifugation using Histopaque®-1077 and cultured in RPMI (containing 10% FBS) with 10 ng/ml human recombinant GM-CSF at 37 °C for 7 days. These cells are confirmed as human macrophages. These macrophages were transfected with vector control, miR-466l,anti-miR-negative or anti-miR-466l (37 °C, 72h). After transfection, total RNA was extracted for reverse transcription. The product cDNA was collected for real-time PCR with respective primers.

ORGANISM(S): Homo sapiens

PROVIDER: GSE52174 | GEO | 2013/11/08

SECONDARY ACCESSION(S): PRJNA227212

REPOSITORIES: GEO

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Expression profile of select inflammation-related genes after miR-466l overexpression or knockdown in human macrophages

Project description:Human Peripheral blood mononuclear cells (PBMC) were isolated from healthy human whole blood by density gradient centrifugation using Histopaque®-1077 and cultured in RPMI (containing 10% FBS) with 10 ng/ml human recombinant GM-CSF at 37 °C for 7 days. These cells are confirmed as human macrophages. These macrophages were transfected with vector control, miR-466l,anti-miR-negative or anti-miR-466l (37 °C, 72h). After transfection, total RNA was extracted for reverse transcription. The product cDNA was collected for real-time PCR with respective primers. In the study presented here, lentiviral vector control (VC), miR-466l, anti-miR-negative (anti-miR-neg) or anti-miR-466l transfected human macrophages were used to acquire expression profiles of a total of 60 unique genes related to inflammation

2013-11-08 | E-GEOD-52174 | biostudies-arrayexpress

Real-time quantitative PCR analysis of human leukemic B-cells.

Project description:Fresh blood samples were collected from 10 CLL (chronic lymphocytic leukemia) previously untreated patients before and after 2 weeks of the first cycle of CCR (cladribine, cyclophosphamide, rituximab) treatment. Peripheral blood mononuclear cells (PBMNCs) were separated from EDTA blood by layering on the Histopaque 1077 and centrifuging on a density gradient. Mean B-cell CD19+ purity was ≥95% as measured by flow cytometry (FC). Microarray analysis was performed using 0.5 microgram of RNA transcribed to cDNA. Prepared cDNA samples were subjected to RT-PCR in duplicate in the TaqMan 7900HT Sequence Detection System (Applied Biosystems).

2013-12-31 | GSE33925 | GEO

Real-time quantitative PCR analysis of human leukemic B-cells [CC treatment]

Project description:Fresh blood samples were collected from 8 CLL (chronic lymphocytic leukemia) previously untreated patients before and after 2 weeks of the first cycle of CC (cladribine, cyclophosphamide) treatment. Peripheral blood mononuclear cells (PBMNCs) were separated from EDTA blood by layering on the Histopaque 1077 and centrifuging on a density gradient. Mean B-cell CD19+ purity was ≥95% as measured by flow cytometry (FC). Microarray analysis was performed using 0.5 microgram of RNA transcribed to cDNA. Prepared cDNA samples were subjected to RT-PCR in duplicate in the TaqMan 7900HT Sequence Detection System (Applied Biosystems).

2015-03-07 | GSE55686 | GEO

Real-time quantitative PCR analysis of human leukemic B-cells.

2013-12-31 | E-GEOD-33925 | biostudies-arrayexpress

The gene expression profile of dendritic cells can be used as a predictor of function.

Project description:The human lung cancer cell line A549 was cultured in media alone, with cyclophosphamide (CPM), oxaliplatin (OXP) or DMSO alone for 72 hours. The exhausted culture media were aspirated and stored at -20C. Peripheral blood mononuclear cells (PBMCs) were isolated from four pathologically healthy donor buffy coats using Histopaque-1077. Following removal of red blood cell and platelet contamination, monocytes were isolated using magnetic beads coated with anti-CD14. Cells were resuspended and cultured in a DC-maturing medium for 7 days. Non- and loosely-adherent cells (the DC fraction) were harvested and purity assessed by CD11c/HLA-DR/CD14 immuno-discrimination by flow cytometry. 1x10^5 unstimulated DCs were cultured with tumour derived supernatants or culture media alone for 24 hours. DCs were then harvested and RNA isolated by Trizol. Total RNA was labelled and hybridised to Illumina Human HT-12v3 arrays. A separate aliquot of DCs from each patient was exposed to supernatant derived from tumour treated with OXP; changes in CD80, CD83 and CD86 were measured and DC populations showing a greater than two-fold up-regulation of these markers were classified as "GOOD_DC".

2011-04-01 | E-MTAB-389 | biostudies-arrayexpress

Transcriptional effects of IL-10 neutralization on LPS-activated M-CSF-derived macrophages

Project description:Analysis of the role of IL-10 on the transcriptional signature of LPS-activated human M-CSF-dependent monocyte-derived macrophages. Methods: Human Peripheral Blood Mononuclear Cells (PBMC) were isolated from buffy coats from donors over a Lymphoprep gradient according to standard procedures. Monocytes were purified from PBMC by magnetic cell sorting using anti-CD14 microbeads (>95% CD14+ cells). Monocytes from three independent donors (0.5 x 106 cells/ml, >95% CD14+ cells) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO2 and 21% O2 for 7 days in the presence of 1000 U/ml M-CSF, with cytokine addition every two days. Then, monocyte-derived macrophages were treated with an anti-IL-10 blocking antibody (anti-IL10) or an isotype-matched antibody (IgG2b) at 2.5 micrograms/ml for 1 hour, and exposed to LPS (10 ng/ml). After 4 hours of LPS treatment, cells were lysed and RNA isolated for transcriptional analysis.

2022-06-08 | GSE181250 | GEO

Real-time quantitative PCR analysis of human leukemic B-cells [CC treatment]

Project description:Fresh blood samples were collected from 8 CLL (chronic lymphocytic leukemia) previously untreated patients before and after 2 weeks of the first cycle of CC (cladribine, cyclophosphamide) treatment. Peripheral blood mononuclear cells (PBMNCs) were separated from EDTA blood by layering on the Histopaque 1077 and centrifuging on a density gradient. Mean B-cell CD19+ purity was ?95% as measured by flow cytometry (FC). Microarray analysis was performed using 0.5 microgram of RNA transcribed to cDNA. Prepared cDNA samples were subjected to RT-PCR in duplicate in the TaqMan 7900HT Sequence Detection System (Applied Biosystems). qPCR gene expression profiling. Intensity of gene expression in CLL cells collected from particular patient before treatment was compared with the gene expression signals in CLL cells from the same patient after 2 weeks of treatment.

2015-03-07 | E-GEOD-55686 | biostudies-arrayexpress

X-ray exposure of peripheral blood mononuclear cells

Project description:Peripheral blood mononuclear cells (PBMCs) were isolated using Histpaque-1077 (Sigma-Aldrich, Belgium) from 10 healthy donors (5 males, 5 females, aged 23-50). Isolated cells were resuspended at a density of 10^6 cells/ml and allowed to equilibrate to culture conditions at 37°C in a humidified 5% CO2 atmosphere. PBMCs were then exposed to 0.0 (sham), 0.1 or 1.0 Gy of X-rays (250 keV - 1 mA, 1 mm Cu) at a dose rate of 0.26 Gy/min. After irradiation, cells were incubated at 37°C in a humidified 5% CO2 atmosphere for 8 h before RNA extraction using the AllPrep DNA/RNA/protein Mini kit (Qiagen). Two weeks later, the experiment was repeated using fresh PBMCs from the same donors, resulting in a total of 60 RNA samples. Total RNA (250 ng) was used for hybridisation to GeneChip Human Gene 1.0 ST arrays (Affymetrix) using the WT expression kit (Ambion), GeneChip Terminal labeling kit (Affymetrix) and the GeneChip Hybridization, Wash and Stain kit (Affymetrix) according to the manufacturer's recommendations.

2016-01-14 | E-MTAB-3463 | biostudies-arrayexpress

Bulk RNA-seq on unstimulated and LPS-stimulated human macrophages

Project description:The goal of this experiment is to profile LPS-stimulated transcriptome changes in human macrophages. Human whole blood was from the Sanofi in-house blood donor service that is approved by the local ethics committee and all blood donors signed informed consent. Human peripheral blood monocytes were isolated from anonymous donors’ prefinal blood using Ficoll density centrifugation, followed by magnetic separation with positive selection (CD14 MicroBeads, Miltenyi Biotec). Monocytes were differentiated into macrophages by culturing in macrophage serum-free medium (Life Technologies) containing 50 ng/ml recombinant human macrophage colony-stimulating factor (Immunotools) for 5 days. Following differentiation, cells were cultured in RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum (Thermo Fisher) and maintained at 37°C in a 5% CO2/air environment. Macrophages were stimulated with 50 ng/ml lipopolysaccharides from Escherichia coli O111:B4 (Sigma) for 24 hours.

2022-03-24 | GSE193336 | GEO

Transcriptional effects of activation stimuli on M-CSF-derived macrophages

Project description:Analysis of the effect of various activation stimuli on the transcriptional signature of human M-CSF-dependent monocyte-derived macrophages. Methods: Human Peripheral Blood Mononuclear Cells (PBMC) were isolated from buffy coats from donors over a Lymphoprep gradient according to standard procedures. Monocytes were purified from PBMC by magnetic cell sorting using anti-CD14 microbeads (>95% CD14+ cells). Monocytes from three independent donors (0.5 x 106 cells/ml, >95% CD14+ cells) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO2 and 21% O2 for 7 days in the presence of 10 ng/ml M-CSF, with cytokine addition every two days. After 7 days, macrophages were either untreated (CNT) or treated with 10 ng/ml E. coli 055:B5 lipopolysaccharide (LPS), 100 ng/ml of the TLR7 agonist CL264 (CL), BSA or 200 mM palmitate (PAL) for 0.5h, 2h, 4h or 12h.

2020-12-09 | GSE156921 | GEO

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