Cells were treated at 37oC for 4 hours with 20uM of PGJ2 (Cayman Chemical Co., Ann Arbor, Michigan) in DMSO. The drug was added drop wise directly into serum-containing medium with a gentle swirl of the culture plate. At the end of the indicated incubation times, the cultures were washed twice with PBS and the cells were harvested. Cell washes removed unattached cells, hence subsequent assays were performed on adherent cells only.
Growth protocol
Human neuroblastoma SK-N-SH cells were maintained at 37oC and 5% CO2 in minimal essential media (MEM) with Eagle's salts containing 2mM L-glutamine, 1mM sodium pyruvate, 0.4% MEM vitamins, 0.4% MEM nonessential amino acids, 100 units/ml penicillin, 100μg/ml streptomycin and 5% normal fetal bovine serum. These cells are derived from peripheral tissue.
Cells were treated at 37oC for 4 hours with vehicle (0.5% DMSO). The drug was added drop wise directly into serum-containing medium with a gentle swirl of the culture plate. At the end of the indicated incubation times, the cultures were washed twice with PBS and the cells were harvested. Cell washes removed unattached cells, hence subsequent assays were performed on adherent cells only.
Growth protocol
Human neuroblastoma SK-N-SH cells were maintained at 37oC and 5% CO2 in minimal essential media (MEM) with Eagle's salts containing 2mM L-glutamine, 1mM sodium pyruvate, 0.4% MEM vitamins, 0.4% MEM nonessential amino acids, 100 units/ml penicillin, 100μg/ml streptomycin and 5% normal fetal bovine serum. These cells are derived from peripheral tissue.
Scanned with a GenePix 4000B scanner, and gridded with GenePix Pro 4.0 software
Description
cDNAs were generated from 5ug of total RNA per sample by reverse transcription (RT) for two hours at 42oC using the 3DNA SubmicroTM Oligo Expression Array Detection Kit (Genisphere, Hatfield, PA) and Superscript II (Invitrogen, Carlsbad, CA). For each microarray chip two consecutive hybridizations were performed with cDNAs and the fluorescent probes following the manufacture’s specifications. Briefly, in the first hybridization reaction the solution containing the mixture of cDNAs obtained from control and treated cells was loaded onto the CAG Human 19K array and incubated overnight at 55oC in a hybridization chamber (Genemachines, San Carlos, CA). The human 19K array (www.cag.icph.org/microarray_facility.htm) contains 60-mer oligonucleotides (Compugen, San Jose, CA) representing 18,861 human genes spotted onto poly-l-lysine coated glass microscope slides. In the second hybridization reaction Cy3 (green) and Cy5 (red) fluorescent probes were used to label the cDNAs from control and PGJ2-treated cells, respectively. The second hybridization was carried out for 3 hours at 65oC in the hybridization chambers. The arrays were scanned using an Axon GenePix 4000B Scanner (Molecular Devices, Sunnyvale, CA) with laser intensities adjusted to bring the brightest spot just below saturation and the data extracted with the Axon GenePix Pro software.
Data processing
The data was filtered so that only spots with a signal intensity of two-fold above background in at least one channel, flagged good (0) and with a diameter above 70 were given a relative expression value: PGJ2-treated vs. control. The data was then normalized globally so the median log ratios for each experiment were equal to zero.
