To identify and isolate HTR-8/SVneo SP and NSP cells, cultured cells were dislodged from dishes with trypsin and EDTA, washed, and suspended at a concentration of 1x 106 cells/mL in RPMI1640 containing 10% FBS, one mM Hepes and five mM EDTA . The cells were labeled in the same medium at 37℃ for 90 minutes with 2.5 μg/mL Hoechst 33342 dye (Moleular Probes Inc., Eugene, Oregon), either alone or in combination with 50 μmol/L verapamil (SIGMA-Aldrich, St.Louis, Missouri). Finally, the cells were counterstained with one μg/mL propidium iodide (PI) to label dead cells. The cells were then analyzed in a FACS Vantage fluorescence-activated cell sorter (BD Bioscience, San Jose, California) using dual wavelength analysis (blue, 424 - 444nm; red 675 nm) after excitation with 350 nm UV light. PI-positive dead cells were excluded from the analysis.
Growth protocol
Parental and NSP cells were cultured in RPMI1640 containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin (HTR-8/SVneo basal medium–[HBM]) providing differentiation conditions. To maintain the SP fraction, SP cells were cultured in 50% HBM and 50% MEF conditioned medium containing one μg heparin/mL and 25 ng /mL FGF2 ( HTR-8/SVneo SP medium [HSM]).
Extracted molecule
total RNA
Extraction protocol
RNA was isolated with Qiagen RNeasy Plus Mini kit (Qiagen, Hilden, Germany) as directed by the manufacturer. 5μg of total RNA was reverse-transcribed to cDNA using oligo dT primer and Superscript Ⅱreverse-transcriptase (Invitrogen, Carlsbad USA).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
1.65 ug of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55ul containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x GEx Hybridization buffer HI-RPM (Agilent) was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays 4x44k (G4112F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then rinsed with Acetonitrile for cleaning up and drying.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505A) using one color scan setting for 4x44k array slides.
Description
gene expression of SP cells cultured for ａ week in HＢＭ
Data processing
The scanned images were analyzed with Feature Extraction Software 9.5.3.1 (Agilent) using default parameters (protocol GE1-v5_95_Feb07 and Grid:026652_D_F_20110819) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
