The SV40-immortalized human urothelial cell line HUC-BC (ATCC) was infected with the retrovirus pLEGFP (Clontech, Mountain View, CA) and cells stably expressing green fluorescent protein (GFP) were selected with flow cytometry.
Treatment protocol
Small intestine submucosa (SIS) membranes encased in plastic inserts (Cook Biotech, West Lafayette, IN) were equilibrated with 3 washes of Hanks Balanced Salt Solution (Invitrogen – Gibco, Carlsbad, CA). After washing, the SIS inserts were incubated in Ham’s F12 ( Invitrogen – Gibco, Carlsbad, CA) with 10% FCS (Invitrogen – Gibco, Carlsbad, CA) for at least 3 hours before cells were placed on membranes. Each SIS insert was placed in an individual well of a 6-well plate (BD Falcon, Bedford, MA) with care taken to ensure air is not captured under the membrane and full contact with the Ham’s F-12 media is made. All but 2.5 ml of the media removed as well as the media on top of the membrane itself. GFP expressing HUC–BC cells were typsinized and brought up in media at a concentration of 150,000 cells/50µl. 50µl seeded onto a membrane surface with area of 50.3 mm2, This cell number is sufficient to cover the membrane with a monolayer of cells and volume enough to keep cells within the membrane insert.
Growth protocol
Cells were grown for 7 days in Ham's F-12 medium containing 10% FCS changed every 2 or 3 days. At day 6, cells were trypsinized from one membrane to obtain a cell number so that the concentration of Enterococcus bacteria would have a multiplicity of infection (MOI) of 10. W32944 strain of Enterococcus was layered onto each Huc-BC/SIS membrane and incubations of 0 minutes, 30 minutes, 60 minutes, 90 minutes, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, and 24 hours were carried out at 37° C in a 5% CO2 incubator.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated at each time point with RNAeasy kit (QIAGEN Inc.,Valencia, CA) according to manufacturer’s protocol.
Label
Cy3
Label protocol
Omniscript reverse-transcriptase, 37 C, 2 hrs
Hybridization protocol
Ventana Discovery 9h, 58 C washed to 0.1xSSC
Scan protocol
scanned on Agilient G2505B scanner Imagene 5.0
Description
Details about microarray handling/processing techniques can be obtained from platform’s manufacturer.
Data processing
Data were normalized as was described previously in Dozmorov I et al., Nucleic Acids Res. 2004 Oct 28;32(19). In general, intensities were converted to expression and normalized to low-expressed genes, which provide possible technical noise level. “Control” and “Blank” spots were excluded prior normalization to exclude part of technical noise. The arrays are then adjusted to each other by robust linear regression and this data presented for each sample. Group of low-expressed genes is selected by normal distribution fitting. This group provides internal standard of measurement noise (ISMN). Genes above 3SD from mean of ISMN were selected as hypervariable yielding 1409 genes. Further filtering by gene variability across time points was done, sorting out genes with standard deviation less than 0.2. This produced 239 genes.
