cell type: lymphoblastoid cell line phenotype: resistant to thiopurine donor: healthy adult
Treatment protocol
Drug concentrations for assessing growth inhibition were 2μM of 6-MP or 5μM of azathioprine, as these concentrations were closed to the mean IC50 and allowed to classify at best the LCLs. Cells were diluted to 200,000 cells/mL and incubated in 96-well plates (Falcon, NY, USA) in a volume of 200μL (40,000 cells/well) for 3 days, with drug working solutions added as needed in three replicates, and six replicates for the controls (cells treated with 20μL PBS). After 72 h, a tetrazolium derivative, MTS reagent (CellTiter 96® AQueous One Solution Cell Proliferation Assay [Promega, WI, USA]), was added (volume 40μL) to each well, including blank wells containing only PBS solution. After further incubation for 4 h, the absorption at 490nm was measured using a microplate reader spectrophotometer (Safir™ [Tecan, Switzerland]), which is reliable to the living cells present in the medium. Growth inhibition relative to control was assessed for each cell line at least two times on two different batches, i.e., different cell vials thawed from liquid nitrogen stock. Reproducibility for repeated thawing was high, as previously reported.
Growth protocol
Human LCLs were obtained from the National Laboratory for the Genetics of Israeli Populations (NLGIP), a human diversity biobank at Tel-Aviv University, Tel Aviv, Israel. The cell lines in the NLGIP collection were generated from lymphocytes isolated from blood samples donated for this purpose by consenting healthy adults (health status, age and ethnicity were self-reported by donors). All cell lines were prepared with EBV generated from the same source of B-95-8 marmoset cell line as described. LCLs were cultured in RPMI medium supplemented with 10% fetal calf serum, antibiotics (100 U/ml penicillin; 100 μg/ml streptomycin), and 2 mM extra l-glutamine (final concentration of 4 mM l-glutamine). Cells were incubated at a temperature of 37°C in a humidified CO2 incubator (6% CO2; 100% humidity) in upright-standing T-75 flasks. The volume was adjusted three-times per week by adding fresh medium to maintain concentrations of between 1 and 2 million cells per ml. Under these growth conditions, cultured LCLs can be maintained continuously for several months in the same flask. Experiments were carried out within 6 months of thawing new vials of each cell line.
Extracted molecule
total RNA
Extraction protocol
Nucleic acid extractions were performed from cells incubated under optimal growth conditions and with no added drugs. Total RNA extractions from cell pellets was performed with the miRNeasy® miniKit (Qiagen, Netherlands) according to the manufacturer's instructions. RNA quality and quantification were assessed using the 2100-Bioanalyzer (Agilent Technologies, CA, USA).
Label
Biotin
Label protocol
Biotinylated cDNA were prepared according to the standard Affymetrix protocol from total RNA (following the GeneChip Whole Transcript (WT) Sense Target Labelling Assay kit, Affymetrix).
Hybridization protocol
Following fragmentation, cDNA were hybridized for 16 hr at 45C on Affymetrix Human Gene 2.0 ST Arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the GCS 3000 scanner (Affymetrix).
Description
9_5452 Healthy adult blood donor from NLGIP (Israel). Thiopurine-resistant lymphoblastoid cell line gene expression data.
Data processing
The data were preprocessed and analyzed using Partek Genomics Suite.
