strain: C57BL/6 cell type: bone marrow stromal cells sample type: ground control gravity type: normal osteo-induced: no
Biomaterial provider
C57BL/6 mice were purchased from Charles River Laboratories (Calco, LC, Italy).
Treatment protocol
Mice between 6 and 8 weeks of age were killed, and bone marrow cells were collected by flushing nucleate cells out of the femurs and tibiae with cold PBS. Cells were cultured (2x106 nucleated cells/10-cm petri dish) in a growth medium [Coon's modified Ham's F12 medium (Biochrom AG, Berlin, Germany)] supplemented with 10% fetal calf serum (GIBCO, Italy), 1% glutamine, 1% penicillin-streptomycin]. No cytokines were added at any stage. Cultures were incubated at 37°C in a 5% CO2 atmosphere. After 3 days, nonadherent cells were removed. When 80% confluent, the adherent cells were trypsinized (0.05% trypsin/EDTA at 37°C for 10 min) and expanded (P1 stage). Cells from the same initial bone marrow sample were cultured at an initial plating density of 1x106 nucleated cells per dish to evaluate their CFU-f (CFU-fibroblast) as a quality control. After 2 weeks of culture, plates were washed with PBS, fixed with 3.7% paraformaldehyde, stained with 1% methylene blue, and colonies were counted. As an additional quality control, we tested BMSC cultures for their ability to undergo differentiation into chondrocytes, osteocytes, and adipocytes.
Growth protocol
Mice were bred and maintained at the Institution's animal facility. The care and use of the animals were in compliance with laws of the Italian Ministry of Health and the guidelines of the European Community.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using RNeasy® MinElute columns (Qiagen,Valencia, CA). RNA concentration and purity were determined from measuring absorbance at 260 and 280 nm; 2 μg total RNA was run on a 1% denaturing gel and 100 ng were loaded on the 2100 Bioanalyzer (Agilent, Palo Alto, CA) to verify RNA integrity.
Label
Biotin
Label protocol
Briefly, accordingly to the recommendations of the manufacturer, 100 ng total RNA was used in the first-round synthesis of double-stranded cDNA. The RNA was reverse transcribed using a WT cDNA synthesis and amplification kit (Affymetrix). The resulting biotin-labeled cRNA was purified using an IVT clean-up kit (Affymetrix) and quantified using a UV spectrophotometer (A260/280; Beckman, Palo Alto, CA). An aliquot (15 µg) of cRNA was fragmented by heat and ion-mediated hydrolysis at 94°C for 35 minutes.
Hybridization protocol
Fragmented cRNA, run on the Bioanalyzer to verify the correct electropherogram, was hybridized in a hybridization oven (16 hours, 45°C) to a Mouse Gene 1.0 ST array (Affymetrix) representing whole-transcript coverage. The washing and staining of the arrays with phycoerythrin-conjugated streptavidin (Molecular Probes, Eugene, OR) was completed in Fluidics Station 450 (Affymetrix).
Scan protocol
The arrays were subsequently scanned using a confocal laser GeneChip Scanner 3000 7G and GeneChip Command Console (Affymetrix).
Description
Bone marrow stromal cells-ground control (bmsc-gc): Cells/skelite constructs, assembled in incubation units (Kayser Italia), maintained in growth medium in manual-operated hardwares (normal gravity) Gene expression data from bone marrow stromal cells - ground control experiment (normal gravity).
