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TK6 and WTK1 LCLs plus NO

ABSTRACT: Cell lines. TK6 and WTK1 are EBV-immortalized human lymphoblastoid cell lines that have been derived from the same donor. TK6 cells bear wild-type TP53 tumor suppressor, while WTK1 cells bear a mutant TP53 containing a C-to-T transition in exon 7, resulting in a change of Met to Ile at codon 237, and coding for a functionally inactive protein. Cells were maintained in exponentially growing suspension culture at 37ºC in a humidified, 5% CO2 atmosphere in RPMI medium1640 supplemented with 10% heat-inactivated calf serum, 100 units/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. Stock cells were subcultured and maintained at a density not greater than 1.5 x 106 cells per ml in 150-mm dishes throughout experiments. NO treatment. Cells at a density of 4 x 105 cells per ml in 100 ml of custom RPMI 1640 medium without calcium nitrate (GIBCO) and calf serum were exposed to pure NO gas (Matheson, Gloucester, MA) by diffusion through Silastic tubing (0.025 in. ID, 0.047 in OD, Dow Corning, Midland, MI) delivery system. NO diffuses through this permeable membrane at a constant rate, and was delivered through 30 cm-long tubing into the medium of well-stirred cell suspensions for 2 h. Cells similarly exposed to argon gas served as negative controls. Total dose (and rate) of NO delivered under these conditions was 390 mmol (533 nM/s), calculated from nitrite plus nitrate content of the medium as determined by automated analysis using the Griess reagent [N-(1-napthy)-ethylenediamine and sulfanilic acid]. At the end of treatment, cells were collected by centrifugation, washed once, resuspended in fresh culture medium containing 10% heat inactivated calf serum, and incubated at 37 ºC. At the indicated times, cells were washed in cold phosphate-buffered saline, harvested and stored at -80 ºC for RNA extraction. cDNA microarray hybridization and analysis. Cells were lysed with Trizol reagent (INVITROGEN, Carlsbad, CA), and total RNA was extracted according to the manufacturer's instructions at 0, 6, 12, and 24h after NO treatment. Fluorescently-labeled cDNA probes were generated using 40 µg of total RNA by a single round of reverse transcription in the presence of aminoallyl-dUTP (SIGMA, St. Louis, MO), followed by a coupling reaction to Cy3 or Cy5 monofunctional NHS-ester (Amersham Pharmacia, Piscataway, NJ). Complex probes (containing untreated (time 0) Cy3-labeled cDNA and NO or argon-treated Cy5-labeled cDNA) were denatured and hybridized to glass slides featuring 9, 180 cDNA clones based on the Hs Unigene build #131 platform (GPL 257), and printed by the NCI Microarray Facility, Advanced Technology Center, Gaithersburg, MD. Upon overnight incubation at 42 ºC, the slides were washed successively in 1 X SSC/ 0.1% SDS, 1 X SSC and 0.2 X SSC for 2 min each, then rinsed in 0.5 X SSC and spin-dried. The two fluorescent intensities were measured simultaneously using a GenePix 4000A scanner, and the acquired image was processed with GenePix Pro 3.0 software (Axon Instruments, Union City, CA). Keywords: time-course

ORGANISM(S): Homo sapiens

PROVIDER: GSE1064 | GEO | 2004/04/01

SECONDARY ACCESSION(S): PRJNA87149

REPOSITORIES: GEO

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Project description:Synechocystis 6803 cells was grown photoautotrophically at 32 °C buffered in BG-11 and bubbled with 3% CO2. A relatively mild Ci stress was applied by switching the aeration from 3% CO2 to air alone. After incubation under designated conditions, a 100-ml aliquot of culture was immediately combined with an equal volume of ice-cold mixture of phenol and ethanol (1:10, w/v) in an ice bath. The resultant cells were collected by centrifugation at 1000 x g for 10 min at 4 °C. Total RNA was isolated with RNeasy Midi Kit (Qiagen, Valencia, CA) and further treated with the DNA-free kit (Ambion, Austin, TX). Fluorescently labeled cDNA was produced via a two-step indirect procedure involving cDNA synthesis from 16 µg of total RNA in a reverse transcriptase reaction incorporating aminoallyl-modified deoxynucleotide, followed by the second step involving chemical coupling of fluorescent dye to the introduced amino moieties of the synthesized cDNA. Labeled cDNA were adjusted to 14.75 µl, and the remainder of the hybridization components containing 2.5 µl of 10 µg µl-1 salmon sperm DNA, 8.75 µl of 20x SSC, 0.25 µl of 10% SDS, and 8.75 µl of formamide were added. The mixture was then heated for 2 min at 99 °C and maintained at 42 °C until hybridization. Hybridizations were preformed in a static incubator at 42 °C for 12-16 h then washed by placing in a 250-ml solution of 2x SSC and 0.1% SDS at 42 °C for 5 min with gentle agitation provided by rotation of a magnetic stir bar. The slide was transferred quickly to a 250-ml solution of 0.1x SSC and 0.1% SDS, incubated for 10 min at room temperature with gentle agitation, and washed five additional times in 0.1x SSC for 1 min at room temperature. Hybridization signals from the microarray were quantified using GenePix Pro 4.1 (Axon Instruments, Union City, CA). The quality control procedures were conducted in the image analysis software, and then data were saved to Acuity 3.1 (Axon Instruments). Keywords: time-course

Project description:Examination of molecular basis of malaria resistance. Spleens from malaria resistant AcB55 and AcB61strains compared with A/J mice. Animals were sacrificed and spleens were rapidly frozen in liquid nitrogen, and subsequently stored at 80oC. Tissues were mechanically homogenized with a polytron and total cellular RNA was extracted using a commercially available reagent (TMTRIzol; Invitrogen). The poly-(A)+ fraction of the RNA was isolated by chromatography with oligo d(T) cellulose beads. For cDNA labeling, either 25 mg of total RNA or 2.5 mg of poly-(A) RNA was converted into cDNA using reverse transcriptase (RT; Super Script II, Invitrogen) and alternatively Cy5 or Cy3-labeled dCTP (1mM, Perkin Elmer-Cetus/NEN, Boston) in a reaction mixture containing 1.5 mL oligo (dT) (100pmol/ mL), 3 mL dNTP-dCTP (6.67mM each), 1 mL dCTP (2mM), 4 mL DTT (100mM), 8 mL 5 X RT Buffer (Invitrogen, California). The reactions were carried out at 42¼C for 3 hrs, after which the RNA was degraded by the addition of 0.5 mL RNase A (1 mg/mL) and 1.5 mL RNaseH 5 units/mL). Labeled cDNA was separated from unincorporated nucleotides (Qiagen column) and further concentrated by evaporation under vacuum. The arrays were pre-hybridized for 1-2hrs with DIGEasy hybridization buffer (Roche) containing 10ug/ml denatured salmon sperm DNA, and 10ug/ml yeast tRNA. The Cy5 and Cy3 labelled cDNAs were combined and hybridized in the same medium and incubated with the arrays for 16-18hrs at 37Co. Finally, the arrays were washed 3 x 10 mins in 0.1 x SSC (20 X SSC is 3M sodium chloride, 0.3 M sodium citrate, pH 7.0), 0.1%SDS at 50Co, and 4 x 3min in 0.1 x SSC at room temperature, and dried by centrifugation. Fluorescent array images were collected for both Cy3 and Cy5 with a ScanArray fluorescent scanner and image intensity data were extracted and analyzed with QuantArray 3.0 analysis software. Quantification data was imported in GeneSpring (Silicon Genetics). The import format was modified to maintain information relevant to user-contributed flagging as well as QC parameter furnished by the array manufacturer. Dye swap and Intensity-based normalization (Lowess) were performed using the default settings. Keywords: other

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Project description:A mouse hepatoma cell line (Hepa1-6 cells, ATCC) was grown in T-25 tissue culture flasks (Corning) at 37ºC in 5% CO2 under two conditions. The first condition was the control, which contained DMEM medium (Gibco BRL) with 25 mM glucose, 4 mM glutamine, 1% penicillin-streptomycin (Sigma), and 10% CPSR-3 (Sigma). The second condition was identical to the first, however the glutamine concentration was altered periodically. Once the cells had grown to confluency, the experiment began at 9:30 PM by exchanging the medium in all flasks. The control flasks received the same medium, containing 4 mM glutamine, while the experimental flasks received identical medium with no glutamine. Twenty-four hours later, the medium was exchanged again, however this time all flasks received medium containing 4 mM glutamine. The first medium exchange was repeated at 48 hours into the experiment, while the second was repeated at 72 hours into the experiment. Hence the glutamine concentration of the experimental flasks oscillated between 0 mM and 4 mM glutamine over 96 hours, while the glutamine concentration of the control flasks remained constant at 4 mM glutamine. At regular intervals, the flasks were sampled. Total RNA was isolated, medium samples were retained, and isotopic measurements were performed from flasks fed different labelled compounds. All samples taken during the experiment were analyzed. The total RNA harvested was labeled and hybridized to DNA microarrays to measure the abundance of transcripts present. Total RNA control samples were labeled using Cy3 dCTP (Perkin-Elmer), and total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was aliquoted into a 6 uL volume. If concentration of the RNA was necessary, this was conducted using a speed vacufuge (Eppendorf) at 60ºC for 10 minutes. The RNA sample was then mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). This reaction mixture was incubated at 42ºC for approximately two hours. Following the reverse transcription reaction, the RNA template was degraded by addition of 2 uL of 1 N NaOH and incubation at 65 ºC for 10 minutes. The alkaline conditions were then neutralized by addition of 2 uL of 1 N HCl. Samples from identical time points that were to be compared on DNA microarrays were then mixed. The combined sample was cleaned to remove extra primers and nucleotides using a QIAquick Nucleotide Removal kit as described by the manufacturer's instructions. Once cleaned, the sample was concentrated using a speed vacufuge (Eppendorf) for 20 minutes at 65ºC. Following concentration, the sample was resuspended in 32 uL of prewarmed hybridization buffer (Clontech Laboratories), 1 uL was removed for quantitation, and hybridized to prepared oligonucleotide microarrays. Hybridization occurred overnight at 55 ºC using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), and then in 1X SSC, and finally 0.1X SSC. Each wash step was conducted for 20 minutes under high agitation. The arrays were dried by centrifugation and scanned using GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and then analyzed using Matlab (The MathWorks, Inc.). The arrays used for hybridization were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). These arrays contained 17,280 features, printed from a synthesized oligonucleotide mouse library (Operon). Briefly, the library was resuspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed using 32 pins (Telechem), at approximately 50% humidity. Once printed, the probes were crosslinked to the glass slides using a UV Stratalinker (Stratagene). The slides were then blocked for 15 minutes in a 250 mL mixture containing 15.7 g/ L succinic anhydride, 239 mL 1-methyl-2-pyrrolidinone, and 10.7 mL of 1 M boric acid, pH 8.0. They were cleaned by rinsing in 250 mL of milli-Q water (X2), followed by 250 mL of 95% ethanol. They were finally dried using filtered air and stored in the dark at room temperature. Keywords = mouse, liver, hepatoma, cell culture, glutamine, carbon metabolism Keywords: time-course

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TK6 and WTK1 LCLs plus NO

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