BM was plated in 600w glucose DMEM, 40% MCDB-201 containing 10ng/mL mEGF, hPDGF-BB and 1000 units/ml mouse leukemia inhibitory factor (LIF), 2creened fetal calf serum (FCS), 1x selenium-insulin-transferrin-ethanolamine (SITE), 0.2 mg/mL linoleic acid-bovine serum albumin (LA-BSA), 0.8 mg/mL bovine serum albumin, 1x chemically defined lipid concentrate and 1x 2-mercaptoethanol, 100 units of penicillin, 1,000 units of streptomycin in a humidified, 502 and 6% CO2, 37oC incubator. After 4 weeks, CD45+ and Ter119+ cells were depleted using a MACS separation CS column, and cells replated at 10 cells/well. Expansion was done by trypsinizing (0.05% trypsin) the cells and replating them every two days at a density of 100-200 cells/cm2. Cells at population doubling >80 were used for gene arrays.
Total RNA was extracted using the RNAeasy microkit (Qiagen, Valencia, CA), DNAse treatment was performed on column
100 ng of total cellular RNA was double amplified and labeled with the Two-Cycle Target Labeling and Control Reagents kit P/N 900494 (Affymetrix Inc., Santa Clara, CA) following the manufacturers' protocol.
Sample was hybridized to Affymetrix mouse 430 2.0 chips, washed, and scanned at the University of Minnesota Affymetrix Microarray Core Facility as described in the Affymetrix GeneChip Expression Analysis Technical Manual. Protocol EukGE-WS2v5_450 .
Array was scanned at the University of Minnesota Affymetrix Microarray Core Facility as described in the Affymetrix GeneChip Expression Analysis Technical Manual. Pixel Size 1.56. Filter 570. Scanner ID 50209060. Number of Scans 1. Scanner Type M10
mouse clone 3, bone marrow 1, biological replicate A, Affymetrix
CEL files were loaded into GeneData Expressionist Refiner (GeneData, San Francisco, CA) to assess overall quality and obtain condensed single intensity values per probeset using the Microarray Analysis Suite Statistical algorithm (MAS 5.0). The mean of mean intensity values for all chips (192) was used to normalize the mean intensity of each chip. Normalization Target: 192, Tau: 0.015, Alpha1: 0.05, Alpha2: 0.065