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Responses of HUVEC to Hypoxia and Reoxygenation

ABSTRACT: Human umbilical vein endothelial cells (HUVEC) were cultivated on 1%-gelatin-coated 75-cm2 culture flasks in Medium 200 supplemented with 2% fetal calf serum, penicillin (100 units/ml), streptomycin (100 units/ml) and Funizone (0.25 μg/ml) supplied by Cascade Biologics (Portland, OR). Prior to an experiment, HUVECs were subcultivated with Trypsin/EDTA onto 1% gelatin coated glass 30-mm glass coverslips in 60 mm glass Petri dishes. The hypoxic treatment was performed in airtight chambers from Mitsubishi Gas Chemical Co. Inc (Remel Scientific, Baton Rouge, LA) Preconditioning consisted of 1-hour hypoxia followed by various periods of reoxygenation (0, 3, 5, 12 and 24 hrs) for gene expression analysis. Total RNA was extracted from cultured HUVECs with TRITM reagent according to the manufacture’s instructions (Molecular Research Center, Cincinnati, OH). Cy5 and Cy3 labeled cDNA probes were generated from total RNA by reverse transcription in the presence of aminoallyl-dUTP according to the method described by Xiang, et al,15. Human universal reference RNA (Stratagene, La Jolla, CA) was Cy3-labeled and used as reference RNA and RNA isolated from HUVEC at 0, 3, 5, 12 and 24 hour following reoxygenation were Cy5-labeled. The high-resolution scans of hybridized microarrays were acquired with a GenePix scanner 4000B (Axon Instruments, Inc., Union City, CA) and tabulated with GenePix pro 5.1 software. Keywords: time-course

ORGANISM(S): Homo sapiens

PROVIDER: GSE1041 | GEO | 2004/02/18

SECONDARY ACCESSION(S): PRJNA87119

REPOSITORIES: GEO

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