Strain: C57BL/6, Sex: Female, Age: Adult, Tissue: Bone marrow (cultured cells), Cell type: Marrow Stromal Cells obtained from the Tulane University Center for Gene Therapy
Femurs and tibiae (cleaned of all connective tissue) were centrifuged to obtain the bone marrow. The cell pellet was resuspended in complete isolation medium (CIM) consisting of RPMI-1640 supplemented with FCS, HS and PenStrep followed by filtration through a 70 µm nylon mesh filter. The cells were plated in 40 ml CIM in a 175-cm2 flask for 24 hours to obtain adherent murine stromal cells (P0). Non-adherent cells were removed by washing with PBS and fresh CIM is added. CIM was added every 3-4 days for a total of 4 weeks. Lifted cells (trpysin-EDTA) were replated (P1) in CIM and CIM replaced every 3-4 days for 1-2 weeks. Cells were lifted and expanded (P2) at (50 cells/cm2) in culture expansion medium (CEM) consisting of: IMDM (Gibco, BRL) with 2mM L-glutamine (Gibco), 100 U/ml penicillin, 100µg/ml streptomycin, 0.25 ?g/ml amphotericin B (Gibco), 10creened FBS (Atlanta biologicals, Lawrenville, GA, Lot C0105) and 10 0.000000e+00quine serum (Hyclone, Lot AQH24495) which is replaced every 3-4 days for 1-2 weeks. Cells were lifted and expanded (P3). Passage 3 cells were either frozen or further expanded as above. Passage 5 frozen cells were obtained from the Tulane University Center for Gene Therapy and thawed and cultured using culture expansion medium (CEM) and serum as recommended by the same center. Briefly, cells were cultured at a plating density of 100 cells/cm2 in CEM in a humidified, 2102, 5% CO2, 37oC incubator. As recommended, a first passage was done one day after thawing and subsequently cells were passaged every six days. RNA samples for gene arrays were taken three days after passaging with plates seeded at 300 cells/cm2 during three consecutive passages (10 population doublings).
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 marrow stromal cells, 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