RAW264.7 cells were treated with KDO, IFN beta and 8Br
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE11449 | GEO | 2008/06/28
SECONDARY ACCESSION(S): PRJNA106481
REPOSITORIES: GEO
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Project description:RAW 264.7 cells were stimulated with KDO (100 ng/ml), IFN-β (300 pM) and/or 8-Br 100 μM. These ligands were applied individually or in combination with KDO for 60 min and 120 min. Total RNA was extracted using TriPure (Roche) following its protocol. Keywords: designed RAW 264.7 cells were stimulated with KDO (100 ng/ml), IFN-β (300 pM) and/or 8-Br 100 μM. These ligands were applied individually or in combination with KDO for 60 min and 120 min. Total RNA was extracted using TriPure (Roche) following its protocol. The detailed procedure is available at http://www.signaling-gateway.org/data/ProtocolLinks.html (AfCS Procedure Protocol PP00000009). Duplicate experiments were done for each treatment. Oligonucleotide array fabrication and annotation 16 K mouse oligonucleotide arrays were fabricated with 15,631 oligomers of 65-bp or 70-bp long. The oligomers were purchased from Operon and Sigma-Genosys and were inkjet-printed onto glass slides by Agilent Technologies. The list of genes is available through the GEO (Gene Expression Omnibus - http://www.ncbi.nlm.nih.gov/geo) as an accession number GPL254. Gene expression analysis The hybridization experiment and analysis were performed as previously described (Park et al., 2004; Zhu et al., 2006; Zhu et al., 2004). Cy5-labeled cRNA (RNA of ligand treated cells) and Cy3-labeled cRNA (RNA of time matched controls) were hybridized in the array. Dye-swap labeling was performed for each pair of samples. The arrays were scanned with Agilent Scanner G2505A (Agilent Technologies) and image files were extracted with background subtraction and dye-normalization using Agilent G2566AA Extraction Software version A.6.1.1 (Agilent Technologies).
2008-06-28 | E-GEOD-11449 | biostudies-arrayexpress
Project description:Preparation of AMDs. 125 μM Ac4ManNAz were incubated with Raw 264.7 (~ 107 cells/dish) for 24, 48, and 72 h, followed by gentle washing with PBS or complete medium to produce Raw 264.7-Ac4ManNAz. Subsequently, Raw 264.7-Ac4ManNAz (~ 107 cells) were resuspended in PBS containing Alkynyl-Ce6(0.1 μM), ascorbic acid (0.2 μM) and CuSO4 (0.2 μM) and incubated for 1h at 37 °C. Then centrifuged (1000 rpm, 3 min) and washed 3 times with PBS to obtain precursor of AMDs. precursor of AMDs (~ 107 cells/tube, 1.0 ml) was immersed in liquid nitrogen, 24 h later, the frozen tubes were thawed at 37°C, centrifuged at 1500 rpm for 3 min, washed and resuspended by PBS to generate the AMDs (5 x 106 units/tube, 1.0 ml).
2024-03-21 | PXD050063 | Pride
Project description:T. cruzi epimastigotes in the logarithmic growth phase (3×106 cells mL-1) were incubated for 12h with bortezomib (1.8 µM), GNF6702 (2.9 µM) or compound 1 (24 µM), equivalent to 8× the EC50 values of each compound. Controls were incubated in the presence of diluent (DMSO). Cells were harvested by centrifugation (1912g, 15 min, 4 °C) and washed with ice-cold PBS (1912g, 5 min, 4 °C), and finally, the cell pellets were resuspended in 1.5 mL of ice-cold lysis buffer (1 mM EDTA, 1 mM DTT, 100 μM TLCK, and 1× Roche EDTA-free cOmplete protease inhibitor cocktail in 50 mM potassium phosphate buffer, pH 7.4). Cell suspensions were submitted to 3 freeze–thaw cycles in a dry ice/ethanol bath to biologically inactivate the parasites and then lysed using the One ShotTM Cell disruptor (Constant Systems, UK) at 30 kpsi.
2022-02-17 | PXD027524 | Pride
Project description:To identify novel LXR target genes, we conducted transcriptional profiling studies using RAW264.7 cells ectopically expressing LXRalpha Total RNA was isolated from RAW264.7 macrophages ectopically expressing LXRalpha as described in Venkateswaran et al. (2000); PNAS 97, 12097-12102. Cells were cultured with DMSO or GW3965 (1 μM) and LG268 (100 nM). Transcriptional profiling was performed at the UCLA microarray core facility using murine Affymetrix 430 2.0 microarrays.
2009-08-01 | E-GEOD-17088 | biostudies-arrayexpress
Project description:Wild-type vs. mutant on candidate cDNA array: Total RNA was extracted from the whole heads of 13.5 wild-type and mutant embryos using TriPure reagent (Roche) and purified using TriPure reagent was purified using the RNeasy Mini kit (Qiagen). Labelled cDNA was generated from RNA extracted from six pooled wild-type and six pooled mutant samples. Labelled cDNA was synthesised using the 3DNA Array 50 kit (Genisphere) and hybridised to the candidate cDNA array. For the oligo array, labelled cDNA was generated from RNA extracted from six pooled wild type and six pooled mutant samples. Labelled cDNA was generated using the amino-alyll labeling method.
2007-12-14 | E-MEXP-1385 | biostudies-arrayexpress
Project description:NIH-3T3 cells were pretreated for 15 min with either DMSO (mock) or cycloheximide followed by addition of either mock, 100 U/ml IFNalpha or 100 U/ml IFNgamma for 1h. During the last 30 min, 500 µM 4-thiouridine was added to cell culture medium. Total cellular RNA was isolated using Trizol reagent and nascent RNA was purified as described (Dölken et al. RNA 2008) . Three replicates of nascent RNA were analyzed by Affymetrix Mouse Gene ST 1.0 arrays
2013-07-04 | GSE30457 | GEO
Project description:Expression data from NIH-3T3 cells treated with mock, 100 U/ml IFN alpha or 100 U/ml gamma for 1 or 3h on nt-RNA labeled for 30-60 min at different times of interferon treatment Differential gene expression caused by IFN alpha or gamma was analyzed in newly transcribed RNA (nt-RNA) of NIH-3T3 cells treated for 1 or 3 h. RNA was labeled for 30 to 60 min and separated from total cellular RNA (tc-RNA) following Trizol RNA preparation and thiol-specific biotinylation We used microarrays to analyze the effects of IFNalpha and gamma treatment in newly transcribed RNA (nt-RNA) Keywords: time course
2009-09-30 | GSE9975 | GEO
Project description:Human primary macrophages were infected with influenza A virus (H3N2/Udorn strain, HA 256) for 6 hrs or left untreated. Cells were collected and lysed with HEPES lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 % NP-40, pH 7.4) including protease and phosphatase inhibitor cocktails. The cell lysates were centrifuged and the supernatant was collected and the protein content was measured with Bio-Rad DC™ protein assay (Bio-Rad). The proteins were reduced, alkylated, and enzymatically digested in-solution with trypsin. The digestion was stopped by adding FA (final c = 1 %). The samples were centrifuged 9168 x g for 10 min and desalted with Sep-Pak Vac RP C18 cartridges (Waters, MA, USA), following fractionation by strong cation exchange chromatography (SCX). The peptides were separated on a 200 x 4.6 mm, 5 μm, 200 Å PolySULFOETHYL A™ column (PolyLC, USA). The fractions were vacuum centrifuged and desalted as before, following phosphopeptide-enrichment with PHOS-Select™ Iron Affinity Gel (Sigma Aldrich, MO, USA). LC-MS/MS was performed with a Q Exactive hybrid quadrupole-orbitrap tandem mass spectrometer coupled to an EASY-nLC 1000 nanoflow liquid chromatograph (Thermo Fisher Scientific). A 100 μm x 3 cm trap column and a 75 μm x 15 cm analytical column were in-house packed with Magic C18AQ resin (200 Å, 5 μm; Michrom Bioresources). The mobile phases were 2% acetonitrile, 0.2% formic acid (A) and 95% acetonitrile, 0.2% formic acid (B). LC gradient elution condition was 2% B (0 min), 20% B (70 min), 40% B (100 min), and then 100% B (105-110 min), with a flow rate of 300 nl/min. Data dependent acquisition was performed in positive ion mode. MS spectra were acquired from m/z 300 to m/z 2000 at a resolution of 70,000 at m/z 200 with a target value of 1,000,000 and maximum injection time of 120 ms. The 10 most abundant precursor ions of which charge states were 2+ or higher were selected for higher energy collisional dissociation (HCD) with an isolation window of 2 and normalized collision energy of 30. MS/MS spectra were acquired at a resolution of 17,500 at m/z 200 with a target value of 50,000, maximum injection time of 250 ms, and the lowest mass fixed at m/z 100. Dynamic exclusion duration was 30 s.
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Project description:The aim of this study was to explore whether mitochondrial- and histone stress cause any common changes in global gene expression. For this experiment, we performed RNA sequencing from control RNAi, cco-1 RNAi (mitochondrial stress) and his-3 RNAi (histone stress) treated C. elegans. Nematodes were collected at L4 larval stage, and total RNA was extracted with TriPure Isolation Reagent (Roche). An Illumina Truseq stranded mRNA library was prepared for nine samples (three biological replicates for each sample). Single-end sequencing with 100 cycles was performed by running the nine samples on one lane of an Illumina Hiseq 2500, yielding a minimum of 26 million reads per sample. Although the two stressors were found to have divergent effects on global gene expression, some biological processes such as innate immunity, heat shock response and chromatin remodeler expression are commonly upregulated upon both mitochondrial- and histone stress.
2017-11-28 | GSE105036 | GEO
Project description:Macrophages are important immune cells operating at the forefront of innate immunity by taking up foreign particles and microbes through phagocytosis. The RAW 264.7 cell line is commonly used for experiments in the macrophage and phagocytosis field. However, little is known how its functions compare to primary macrophages. Here, we have performed an in-depth proteomics characterisation of phagosomes from RAW 264.7 and bone marrow-derived macrophages by quantifying more than 2500 phagosomal proteins. Our data indicates that there are significant differences for a large number of proteins including important receptors such as mannose receptor 1 and Siglec-1.
2020-02-08 | MSV000084938 | MassIVE