Iron chelation on human cytomegalovirus infected MRC-5 fibroblasts

ABSTRACT: MRC-5 cells (American Type Culture Collection, Manassas, VA) derived from human lung fibroblasts (passages 19-25) were cultured in MEM with Earle’s salts, supplemented with 2 mM glutamine and 10% heat-inactivated FCS. Cells were grown in an incubator with a humidified atmosphere of 5% CO2 in air at 37oC for several days to reach confluence. HCMV strain AD169 (American Type Culture Collection, Manassas, VA) was used in these studies. A continually replenished stock of HCMV was cultured in confluent MRC-5 cells. At the appropriate time, the infected cells were lysed and the infectivity of the resultant viral stock was assayed by plaque assay. For the studies reported here, HCMV infection was carried out by exposing the confluent MRC-5 cells to a virus stock at a multiplicity of infection (MOI) of ~3-5 plaque-forming units (PFU) per cell for 1 h at 370C. To control for non-specific effects of the cell lysate portion of the viral stock, a parallel set of MRC-5 cells were always mock infected by a 1h exposure to a lysate of uninfected MRC-5 cells. After the infectious period of exposure, both mock- and virus-treated cells were rinsed with phosphate buffered saline (PBS) followed by fresh culture medium and returned to cell culture for various post-exposure (PE) times. Mock- or HCMV-infected cells were homogenized in guanidinium isothiocyanate, followed by the isolation of total RNA following the method of Chirgwin et al. (PubMed ID 518835). Purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis prior to use. Two successive oligo (dT) cellulose columns were then used to isolate poly A+ RNA for microarray analysis, yielding 600 ng of mRNA per channel (mock-infected or HCMV-infected cells). These were labeled with Cy3 and Cy5 dyes respectively prior to hybridization with a UniGEM V cDNA microarray (Incyte Genomics, St Louis, MO). Keywords: other

ORGANISM(S): Homo sapiens

PROVIDER: GSE672 | GEO | 2003/09/20

SECONDARY ACCESSION(S): PRJNA87611

REPOSITORIES: GEO