GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Series GSE1064

Query DataSets for GSE1064

Status

Public on Apr 01, 2004

Title

TK6 and WTK1 LCLs plus NO

Organism

Homo sapiens

Experiment type

Expression profiling by array

Summary

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

Citation(s)

15126337

Submission date

Feb 25, 2004

Last update date

Mar 02, 2012

Contact name

Ana I Robles

E-mail(s)

Ana_Robles@nih.gov

Phone

301-496-1729