HIGK cells were originally obtained by transfection of primary gingival epithelial cells with E6/E7 from HPV (Oda et al., 1990). HIGK cells were reported to be stable for over 350 passages (Oda et al., 1996). They were originally obtained at passage number 82, and used by passage number 90 for the the experiments described herein.
Treatment protocol
Bacteria at mid-log phase were harvested and washed by centrifugation, and resuspended in antibiotic-free K-SFM media. HIGK cells (10e7) were co-cultured with bacteria to give a total association (adhered plus invaded) of approximately 100 organisms per epithelial cell. Numbers of adhered and invaded organisms were confirmed in parallel experiments by plate counting. After 2 hours at 37C in 5% CO2, the HIGK cells were lysed with Trizol (Invitrogen) prior to RNA extraction.
Growth protocol
HIGK cells were cultured under 5% CO2 in keratinocyte serum-free medium (K-SFM, Gibco/Invitrogen, Carlsbad, CA) supplemented with: 0.05 mM calcium chloride, 200 mM L-glutamine (Gibco/Invitrogen, Carlsbad, CA).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted, DNAse-treated, purified and quantified according to standard methods (Qiagen and Affymetrix).
Label
biotin
Label protocol
cRNA synthesis was performed with 10 ug of total cellular RNA, based on the Affymetrix protocol. Double-stranded cDNA was synthesized according to a standardized protocol (SuperScript doublestranded cDNA synthesis kit; Invitrogen, Carlsbad, CA). cRNA was transcribed in vitro, incorporating biotinylated nucleotides by using a BioArray high-yield RNA transcript labeling kit (T7) (Enzo Life Sciences, Farmingdale, NY).
Hybridization protocol
Following fragmentation, human HG U133-A oligonucleotide microarrays (Affymetrix) were hybridized for 16 h at 45C, stained with phycoerythrin-conjugated streptavidin and washed according to the Affymetrix protocol (EukGE-WS2v4).
Scan protocol
GeneChips were scanned with an Affymetrix fluidics station, and scanned with an Affymetrix GeneChip 3000 scanner.
Description
Human Immortalized Gingival Keratinocyte (Oda, D., and Watson, E. (1990) Human oral epithelial cell culture. Improved conditions for reproducible culture in serum-free medium. In Vitro Cell Dev Biol Anim 26: 589–595. Oda, D., Bigler, L., Lee, P., and Blanton, R. (1996) HPV immortalization of human oral epithelial cells: a model for carcinogenesis. Exp Cell Res 226: 164–169.)
Data processing
The data were analyzed with Affymetrix GCOS using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.
