cell type: BM stroma prepared from BM aspirate of healthy donor treatment: Vehicle
Growth protocol
Stroma was prepared from BM aspirates of healthy donors, 18 to 25 years of age. Unfractionated cells from BM aspirates were cultured at 37°C in α-MEM with 12.5% FCS, 12.5% horse serum, 0.1 μM hydrocortisone, 0.1 mM 2-ME, and 1.6 mM glutamine. At day 3, red blood cells and granulocytes were eliminated by Ficoll-Hypaque density gradient and the mononuclear fraction replaced into the tissue culture flask. Cultures were reincubated with weekly replacement of 50% medium until confluence.
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using Trizol following manufacturer's instructions
Label
Cy3
Label protocol
Total RNA was used to prepare cDNA probes. cDNA probes were labeled with biotin and DNP for control and stimulated stroma respectively. Total RNA, control RNA, and biotin or DNP reaction mix were mixed and incubated at 65 °C for 10 minutes, cooled to 25 °C for 5 minutes, and warmed to 42 °C. After addition of 10X RT reaction buffer and AMV RT/RNase inhibitor mix, the samples were reverse transcribed at 42 °C for 1 h, denatured at 95 °C for 5 minutes, and snap cooled on ice. 1N NaOH was added, followed by incubation at 37 °C for 10 minutes. 1N HCl was added, followed by 3M Na Acetate, 100% ethanol. Following precipitation, samples were washed with 70% and 95% ethanol, dried, and resuspended in 1X TE (10 mM Tris, 1 mM EDTA, pH 7.5).
cell type: BM stroma prepared from BM aspirate of healthy donor treatment: 10 nM Substance P
Growth protocol
Stroma was prepared from BM aspirates of healthy donors, 18 to 25 years of age. Unfractionated cells from BM aspirates were cultured at 37°C in α-MEM with 12.5% FCS, 12.5% horse serum, 0.1 μM hydrocortisone, 0.1 mM 2-ME, and 1.6 mM glutamine. At day 3, red blood cells and granulocytes were eliminated by Ficoll-Hypaque density gradient and the mononuclear fraction replaced into the tissue culture flask. Cultures were reincubated with weekly replacement of 50% medium until confluence.
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using Trizol following manufacturer's instructions
Label
Cy5
Label protocol
Total RNA was used to prepare cDNA probes. cDNA probes were labeled with biotin and DNP for control and stimulated stroma respectively. Total RNA, control RNA, and biotin or DNP reaction mix were mixed and incubated at 65 °C for 10 minutes, cooled to 25 °C for 5 minutes, and warmed to 42 °C. After addition of 10X RT reaction buffer and AMV RT/RNase inhibitor mix, the samples were reverse transcribed at 42 °C for 1 h, denatured at 95 °C for 5 minutes, and snap cooled on ice. 1N NaOH was added, followed by incubation at 37 °C for 10 minutes. 1N HCl was added, followed by 3M Na Acetate, 100% ethanol. Following precipitation, samples were washed with 70% and 95% ethanol, dried, and resuspended in 1X TE (10 mM Tris, 1 mM EDTA, pH 7.5).
Hybridization protocol
Equal amounts of cDNA from control and stimulated BM stroma were added together and dried down. Samples were resuspended and heated to 90 °C for two minutes. Following addition of hybridization buffer, samples were added to cDNA arrays, coverslips were applied, and incubated overnight at 65 °C. Hybrids were detected with the NEN Life Science Tyramide Signal Amplification system.
Scan protocol
Scanned on GSI Lumonics ScanArray 3000. Images were quantified using NEN data analysis, version 2.0.
Description
Paired biological replicate 2 of 3. Control BM stromal cells vs. Substance P treated BM stromal cells. Stromal cells were harvested from the same donor.
Data processing
log2 transformed, background subtracted signal intensity
