Cytotoxic T lymphocyte cell line CTLL-2, derived from a C57BL/6 mouse, dependent upon IL-2 for growth
Biomaterial provider
The American Type Culture Collection (ATCC)
Treatment protocol
Cells were cultured in D10 medium in an incubator with 5% CO2 at 37oC to mid-log phase (~2 X 105 cells/mL), and collected by centrifugation, washed with phosphate buffered saline (PBS) (Invitrogen), resuspended at 1 X 106 cells/mL in the medium without IL-2 (IL-2 starvation) and cultured for 14 hours at 37oC with 5% CO2, followed by treatment with cycloheximide (20 ?g/mL) for 30min at 37oC with 5% CO2.
Growth protocol
Mouse CTLL-2 cells was maintained in an incubator with 5% CO2 at 37oC in D10 medium (D10 = ATCC modified RPMI-1640 medium (10 mM HEPES, 2 mM L-glutamine, 1 mM Sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate), supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 U/mL penicillin, 50 mg/mL streptomycin, 1 mM sodium pyruvate, 45 µM ?-mercaptoethanol), and 50 U/mL human rIL-2 (Chiron).
Extracted molecule
total RNA
Extraction protocol
Cells was collected and washed with PBS, resuspended in PBS before adding RNAlater RNA stabilization solution (Qiagen). Total RNA was isolated using RNeasy Mini Kit (Qiagen) according to manufacturer’s protocol.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 4 ?g total RNA (GeneChip Expression Analysis, Technical Manual (701021 Rev. 5), 2005, Affymetrix).
Hybridization protocol
Following fragmentation, 10 ?g of cRNA were hybridized for 16 hr at 45C on GeneChip Mouse Genome 430 2.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Scan protocol
GeneChips were scanned using Affymetrix Gene-Chip scanner 3000.
Description
Gene expression data from T cells cultured without IL-2 for 14 hours followed by cycloheximide for 30min
Data processing
The data were analyzed with Affymetric GeneChip Operating Software (GCOS, version 1.3) using Affymetrix default analysis settings and global scaling as normalization method.
