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.
The standard deviation calculated for the Cy5 channel mean intensity of the spot.
B635_MEDIAN
Cy5 channel background median intensity around the spot.
B635_MEAN
Cy5 channel background average intensity around the spot.
B635_SD
The standard deviation calculated for the Cy5 channel background average intensity around the spot.
F635_QUALITY
Cy5 channel percentage of pixels in the spot more than one standard deviation above background.
F532_MEDIAN
Cy3 channel median intensity of the spot.
F532_MEAN
Cy3 channel average intensity of the spot.
F532_SD
The standard deviation calculated for the Cy3 channel mean intensity of the spot.
B532_MEDIAN
Cy3 channel background median intensity around the spot.
B532_MEAN
Cy3 channel background average intensity around the spot.
B532_SD
The standard deviation calculated for the Cy3 channel background average intensity around the spot.
F532_QUALITY
Cy3 channel percentage of pixels in the spot more than one standard deviation above background.
FLAGS
The indication of spot quality determined by the image analysis software. Unflaged spot was assigned a value of zero. A negative value was assigned to a spot with intensity near background, uneven spot or background intensity.
UNF_VALUE
log ratio of Median of Ratios between Cy5 and Cy3 channels. (log2 of Median of Ratios). The Median of Ratios is the median of the pixel-by-pixel ratios of pixel intensities that have had the median background intensity subtracted.
