Female, age 56 Disease status: HB (control (stress incontinence, no bladder pain)) Culture medium: DM (differentiating medium)
Treatment protocol
A single cold-cup bladder biopsy was taken and transported immediately to the laboratory in growth medium. Each biopsy was divided into 6-10 pieces using fine scissors and optical loupes. The pieces were distributed into a 6-well plate with 1-2 pieces per well. 1.5 ml growth medium was added to each well and the plate was incubated at 37oC, 5% carbon dioxide. Additional medium was added to the wells on day 3, and a schedule of changing medium three times a week was started on day 5. When the epithelial cells had grown from the primary explants, they were passaged using TrypLE Express (Gibco, Invitrogen Corporation, Carlsbad, CA) according to the manufacturer’s instructions and re-seeded into the appropriate size vessel for a 2500 cells/cm2 density.
Growth protocol
The initial growth medium (KM) was according to Southgate: Keratinocyte Growth Medium with 5 ng/ml recombinant human epidermal growth factor (EGF), 50 ?g/ml bovine pituitary extract (all from Gibco) and 30 ng/ml cholera toxin (Sigma, St. Louis, MO). To induce differentiation, medium was changed two days before extraction to a high-calcium, serum-containing medium (DM) described by Keay: DMEM-F12 (Media Tech, Herndon, VA) with 10% fetal bovine serum , 1% antibiotic/antimycotic solution, 1% L-glutamine, 1.0 U/ml insulin (all from Sigma), and 5 ?g/ml hEGF (R & D Systems, Minneapolis, MN). Total RNA was extracted from cells at passages 3 to 6.
Extracted molecule
total RNA
Extraction protocol
Standard Affymetrix
Label
Standard Affymetrix
Label protocol
Standard Affymetrix
Hybridization protocol
Standard Affimetrix
Scan protocol
Affy
Description
Evaluate gene expression profiles after inducing differentiation in cultured interstitial cystitis (IC) and control urothelial cells.
Data processing
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.
