5 protocols
AccessionType
hybridization
P-AGIL-13
labeling
P-AGIL-12
image_acquisition
The probe mixture was hybridized for 40 hours at 65C to a G4412A Human Genome CGH Microarray Kit 105A (Agilent Technologies, Santa Clara, California). The array was scanned after washing with a G2565BA Microarray Scanner and fluorescent signals were acquired using Feature Extraction software (Agilent Technologies).
(Parameters: Scanning hardware = G2565BA DNA microarray scanner [Agilent], Scanning software = Feature Extraction Software [Agilent])
growth protocol
Human epithelioid sarcoma cell line ESX was established in our laboratory. ESX cells were cultured in Iscove's modified Dulbecco's Eagle's medium with 10% FBS at 37 degrees C in 5% CO2. Passage was performed about twice per week. No special reagents were used prior to ALDEFLUOR assay. The ALDEFLUOR kit (StemCell Technologies, Vancouver, Canada) was used to separate the population with a high and low ALDH1 activity. Cells (1*106) were suspended in ALDEFLUOR assay buffer containing ALDH1 substrate, bodipy-aminoacetaldehyde, at the concentration of 1umol/L and incubated for 50 min at 37? . ALDHhigh and ALDHlow cells were isolated using FACS Aria II (BD Bioscience, San Jose, CA). Propidium isodide was also used to stain live cells. We found that ALDHhigh cells of ESX contained larger proportion of CSCs/CICs showing in vitro and in vivo tumorigenesis more than ALDHlow cells.
nucleic acid extraction protocol
RNA from ALDHhigh cells was labeled with Cy5 dye and RNA from ALDHlow cells were labeled with Cy3 dye. The probe mixture was hybridized for 40 hours at 65C to a G4412A Human Genome CGH Microarray Kit 105A (Agilent Technologies, Santa Clara, California). The array was scanned after washing with a G2565BA Microarray Scanner and fluorescent signals were acquired using Feature Extraction software (Agilent Technologies). The average expression ratio of Cy5 to Cy3 was determined per gene. A dye swap experiment was also done to label ALDHhigh and ALDHlow cells with Cy3 and Cy5, respectively.
(Parameters: Extracted product = total_RNA, Amplification = none)