age (years): 67 gender: m sample source: PB karyotype: normal igvh status: unmutated tp53 mutation: MUT treatment time: 20h treatment: BV6
Primary CLL samples were cultivated using RPMI 1640 (Biochrom AG, Berlin, Germany), supplemented with 10% FCS (Sigma-Aldrich, St. Louis, MO, USA), 2 mM L-Glutamin (Biochrom AG), and Penicillin/Streptomycin (GIBCO, Invitrogen Corporation, Grand Island, NY, USA). Prior to treatment, cells were stained with trypan blue (Sigma-Aldrich), counted, and diluted to a density of 1.0x106 cells/ml. Thawing of viably frozen samples followed the DSMZ (German Collection of microorganisms and cell lines, Braunschweig) guideline. Agents used to treat primary AML sample for 4 and 20 hours in vitro were DMSO (control/carrier; dimethyl sulfoxide; Sigma-Aldrich), and BV6 was synthesized at Genentech, Inc. (South San Francisco, CA, USA).
The total RNA was purified with a TRIzol-based protocol.
For each sample preparation, total RNA was converted into double-stranded cDNA by reverse transcription using the 3' IVT Express Kit from Affymetrix according to the manufacturer's recommendations.
Hybridization, washing, and staining protocols, respectively, were performed on Affymetrix GeneChip instruments (Hybridization Oven 640, Fluidics Station FS450) as recommended by the manufacturer.
Scanning was performed on Affymetrix GeneChip Scanner GCS3000 instruments as recommended by the manufacturer using default settings. The software used was GCOS 1.2 or higher.
The data were analyzed with BRB ArrayTools using RMA. Intensity values were log2 transformed