tissue of origin: Melanoma cell line: M14 epithelial: no ploidy: 3n+/-, Near-triploid 69+/- (58-80) p53 mutation: MT doubling time: 26.3 contributing institute: John Wayne Cancer Clinic, UCLA School of Medicine contributing person: D.H. Kern, John Wayne Cancer Clinic, UCLA School of Medicine reference: Cancer Res 48: 578-582, 1988
Treatment protocol
No treatment
Growth protocol
Cells grown in RPMI 1640 with 5% FBS (Bio Whittaker, not heat inactivated) and 1% L-glutamine. Cells not grown past 80% confluencey for attached cells, or 0,5 x 10-6 cells for suspended. Trypsinize (for attached cells) cells with 5 ml trypsin-EDTA per T162, for 15 min., at 370C. Spin down suspended cells, and resuspend in ~3ml growth media. Count cells. Pass 1x10-6 cells into new T162’s w 30 ml media. Repeat growth cycle until desired amount of cells are available. Cells are not grown past passage 20.
Extracted molecule
total RNA
Extraction protocol
Growth schedule prior to harvest: Grow cells to ~80 confluencey. Re-feed cells the day prior to harvest. Draw off media from attached cells, or spin down suspended cells. Rinse cells with ~10 ml 1x PBS. Purify using Quiagen Midi Kit (Cat # 75144) per instructions. In brief, lyse cells in 15 ml lysis buffer (w 10 ul fresh BME per ml) per 4 T162s. Scrape (attached) cells. Pipet into a 50 ml tube. Repeat with the next set of 4 flasks. When done, vortex 10 sec.. Draw lysate thru a 20 guage needle 12x’s. Freeze lysate at -80 deg C. Use a maximum of 100 x 10-6 cells per column.
Label
biotin
Label protocol
Done by Genelogic according to manufacturer's recommendations
Hybridization protocol
Done by Genelogic according to manufacturer's recommendations
Scan protocol
Affymetrix MicroArraySuite
Description
Melanotic melanoma
Data processing
Probes were normalized using RMA in Partek Genomics Suite version 6.2
