Chronic lymphocytic leukemia cells are activated and proliferate in response to specific Th cells
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ABSTRACT: Changes in gene expression profile of CLL cells in response to an antigen-specific Th cell clone that is specific for a Ckappa peptide of mouse Abs. Mouse anti human BCR mAbs were used to ligate BCR and deliver antigen to CLL cells. 32-45 x10e6 PBMC were incubated overnight with or without 2 μg/ml of Ms κ+IgG anti-kappa and anti-lambda mAbs. The PBMC were then cultured in presence or absence of 12.5 x10e6 T18 cells. On day 3, CLL cells were purified by negative selection using CD3 and CD14 Dynabeads (Invitrogen). RNA from CLL cells was controlled and quantified on a NanoDrop1000 (Thermo Scientific, Wilmington, DE, USA). 290ng per sample was amplified and labeled with the TotalPrep™-96 RNA Amplification Kit (Illumina, San Diego, CA, USA). Sample quality was further tested by Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA). 1500ng of biotin labeled cRNA was used to hybridize onto Illumina HumanWG-6 v3 Expression BeadChips. After scanning, the results were imported into Illumina GenomeStudio v. 2010.1, Gene Expression module v. 1.6.0 for quality control and data export. Analysis was performed utilizing GeneSpring GX v11 software (Agilent, Santa Clara, CA, USA). In GeneSpring GX, Logbase 2-transformed data were normalized to the 75 percentile with baseline transformation to median of all samples. Flag Information: Lower cutoff for 'Present' call: 0.8, Upper cutoff for 'Absent' call: 0.6
Project description:Changes in gene expression profile of CLL cells in response to an antigen-specific Th cell clone that is specific for a Ckappa peptide of mouse Abs. Mouse anti human BCR mAbs were used to ligate BCR and deliver antigen to CLL cells. 32-45 x10e6 PBMC were incubated overnight with or without 2 μg/ml of Ms κ+IgG anti-kappa and anti-lambda mAbs. The PBMC were then cultured in presence or absence of 12.5 x10e6 T18 cells. On day 3, CLL cells were purified by negative selection using CD3 and CD14 Dynabeads (Invitrogen). RNA from CLL cells was controlled and quantified on a NanoDrop1000 (Thermo Scientific, Wilmington, DE, USA). 290ng per sample was amplified and labeled with the TotalPrep™-96 RNA Amplification Kit (Illumina, San Diego, CA, USA). Sample quality was further tested by Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA). 1500ng of biotin labeled cRNA was used to hybridize onto Illumina HumanWG-6 v3 Expression BeadChips. After scanning, the results were imported into Illumina GenomeStudio v. 2010.1, Gene Expression module v. 1.6.0 for quality control and data export. Analysis was performed utilizing GeneSpring GX v11 software (Agilent, Santa Clara, CA, USA). In GeneSpring GX, Logbase 2-transformed data were normalized to the 75 percentile with baseline transformation to median of all samples. Flag Information: Lower cutoff for 'Present' call: 0.8, Upper cutoff for 'Absent' call: 0.6 Testing antigen dependent Th cell - CLL cell collaboration, effects on gene expression of CLL cells
Project description:Systemic lupus erythematosus (SLE) is a chronic, autoimmune disease affecting multiple heterogeneous organs and systems. SLE is associated with increased risk of atherosclerosis and increased cardiovascular complications. In this study, we specifically aimed to identify patients with SLE who are genetically at risk for developing atherosclerosis. Sureprint G3 Human Gene Expression 8x60K Microarray kit (Agilent technologies, Santa Clara, CA, USA) was used in our study. Genes showing differences in expression between the groups were identified by using GeneSpring GX 10.0 program. A total of 155 genes showing expression level difference were detected between SLE patients and healthy controls. In molecular network analysis.
Project description:Stat5+/- mice were bred into the C57BL/6 background. Stat5+/- mice were intercrossed and mouse embryonic fibroblasts (MEFs) were isolated from 12.5-13.5-day WT or Stat5-/- fetuses. The retroviral-expression vector carrying a wild-type Stat5A gene based on an MSCV-IRES-GFP backbone (gift from Richard Moriggl, Ludwig-Boltzmann Institute, Vienna, Austria) was infected into Stat5-/- MEFs. FACS was used to select GFP+ cells. After 5 hours starvation in serum free medium with 0.1% of BSA, MEFs were treated with growth hormone for 2 hours. Total cellular RNA from each group of the MEFs was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Microarray analyses were performed using Affymetrix Mouse Genome 430 2.0 GeneChips (Affymetrix, Santa Clara, CA) (six groups, biological replicates for each group). Expression values were determined with GeneChip Operating Software (GCOS) v1.1.1 software. RMA signals were summarized using GeneSpring GX 10.0.1 (Agilent) and normalized by quantile normalization. All data analysis was performed with GeneSpring software GX 10.01.
Project description:For the microarray experiments, MV4-11 and MOLM-14 cells were treated with DMSO control, ABT-869 3 nM, SAHA 6 uM and combination therapy for 24 hours. Cells were then washed in PBS and high-quality total RNA was extracted RNeasy Midi Kit, according to the manufacturer’s instruction (Qiagen, Valencia, USA). RNA quantity, quality, and purity were assessed with the use of the RNA 6000 Nano assay on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara CA, USA). Gene expression profiling was performed using Affymetric U133plus2.0 gene chip (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s protocol.
Project description:Stat5+/- mice were bred into the C57BL/6 background. Stat5+/- mice were intercrossed and mouse embryonic fibroblasts (MEFs) were isolated from 12.5-13.5-day WT or Stat5-/- fetuses. The retroviral-expression vector carrying a DNA binding domain mutant Stat5A (E437/E438?AA) or tyrosine-phosphorylated Stat5A Y694F mutation based on an MSCV-IRES-GFP backbone (gift from Richard Moriggl, Ludwig-Boltzmann Institute, Vienna, Austria) was infected into Stat5-/- MEFs. FACS was used to select GFP+ cells. After 5 hours starvation in serum free medium with 0.1% of BSA, MEFs were treated with growth hormone for 2 hours. Total cellular RNA from each group of the MEFs was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Microarray analyses were performed using Affymetrix Mouse Genome 430 2.0 GeneChips (Affymetrix, Santa Clara, CA) (four groups, biological replicates for each group). Expression values were determined with GeneChip Operating Software (GCOS) v1.1.1 software. RMA signals were summarized using GeneSpring GX 10.0.1 (Agilent) and normalized by quantile normalization. All data analysis was performed with GeneSpring software GX 10.01.
Project description:Mouse livers were analyzed by lipidomics and urine by metabolomics.An Agilent Ultra-Performance Liquid Chromatography/Electrospray Ion Quadrupole Time-of-Flight-Mass Spectrometer (UPLC-ESI-QTOFMS) (Agilent, Santa Clara, CA) was utilized for lipid and other metabolites proofing in this work.
Project description:RNA was isolated from flow-sorted Kupffer cells under the two different conditions (tacrolimus and control) using RNeasy mini kit (Qiagen). Integrity of DNA and RNA was assessed by Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). 500 nanograms of RNA were used for analysis with Affymetrix Mouse Gene 2.0 ST platform
Project description:This study investigated the mechanism by which 300 kDa HA (1 mg/mL) promotes the proliferation of hAECs. P1 hAECs in logarithmic growth phase were harvested, inoculated into 6-well plates at 3×105/well, and cultured with 5% CO2 at 37 °C. The medium was changed after 3 days, and then the cells were cultured for 18 or 36 h in the presence and absence of HA (300 kDa, 1 mg/mL). The total RNA was purified using the RNeasy MinElute kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized using a SuperScript III Reverse Transcriptase Kit (Invitrogen, Shanghai, China). Real-time PCR was conducted, and the 2–∆∆Ct method was used to quantify gene expression levels. The signal was collected by Agilent Feature Extraction software and analyzed by Agilent GeneSpring GX software.
Project description:High quality RNA was extracted from the whole seedlings (Combined root and leaf samples) using TRI Reagent (Ambion, Inc. USA) and pooled from 12 independent stressed and non-stressed plant samples separately, and treated with DNase-I (QIAGEN GmbH, Germany). Subsequently, RNA cleanup was carried out using RNeasy Plant Mini Kit (QIAGEN GmbH, Germany) and 5 ug of total RNA from each sample in triplicates were reverse-transcribed to double stranded cDNA using the GeneChipï¾® One-Cycle cDNA Synthesis Kit. The biotin-labelled cRNA was made using the GeneChipï¾® IVT Labelling Kit (Affymetrix, CA, USA). Twenty microgram of cRNA samples was fragmented and out of which which 7.5 ug cRNA were hybridized for 16 hours at 45C to the Affymetrix GeneChipï¾® Rice Genome Array (Santa Clara, CA, USA). After washing and staining with R-phycoerythrin streptavidin in a Fluidics Station, using the Genechipï¾® Fluidics Station 450, the arrays were scanned by the Genechipï¾® 3000 Scanner. The chip images were scanned and extracted using default settings and the CEL files were produced with the Affymetrix GeneChip Operating Software (GCOS 1.2). The resulting .CEL files were imported into the GeneSpring GX 10 (Agilent Technologies Inc, Santa Clara CA) and normalized with the PLIER16 algorithm. The resulting expression values were log2-transformed. Average log signal intensity values of three technical replicates for each sample were used for advance analysis.
Project description:Tomato pericarp tissues at three different stages (green, breaker and ripening) of fruit development from both the above genotypes were collected using a sterile surgical blade and flash frozen in liquid nitrogen. High quality RNA was extracted from the frozen pericarp tissue using TRI Reagent (Ambion, INC. USA) and pooled at least from three independent samples and treated with DNase-I (QIAGEN GmbH, Germany). RNA cleanup was carried out using RNeasy Plant Mini Kit (QIAGEN GmbH, Germany) and 5ug of total RNA from each sample with three biological replications were reverse-transcribed to double stranded cDNA using the GeneChipR One-Cycle cDNA Synthesis Kit. The biotin-labelled cRNA was made using the GeneChipR IVT Labelling Kit (Affymetrix, CA, USA). 15 ug of cRNA samples was fragmented and was hybridized for 16 hours at 45C to the Affymetrix tomato GeneChip Array (Santa Clara, CA, USA). After washing and staining with R-phycoerythrin streptavidin in a Fluidics Station, using the GenechipR Fluidics Station 450, the arrays were scanned by the GenechipR 3000 Scanner. The chip images were scanned and extracted using default settings and the .CEL files were produced by using the Affymetrix GeneChip Operating Software (GCOS 1.2). Samples that passed through various quality checks (data not shown) were normalized with RMA algorithm using GeneSpring GX 10 (Agilent Technologies Inc., Santa Clara, CA, USA). Probes showing twofold up- or down-regulation (in Log2 transformed values) compared to corresponding control condition were taken only into consideration for further analysis