Hypothermia was initiated by lowering the temperature of the cell culture incubator to 32 degrees celcius. The control arm of the experiment was incubated at 37 degrees celcius. Three dishes per group were incubated for various durations (0h, 0.5h, 1h, 2h, 4h, 8h, 18h).
Growth protocol
Around 1 million primary mouse embryonic fibroblasts (Millipore) were seeded onto 100-mm culture dishes and were grown in DMEM (high glucose 4.5g/l, supplemented with 10% FBS and L-glutamine, PAA) in normal conditions (atmospheric oxygen, 5% CO2 at 37C) until 60-70% convulent.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from cells with Trizol® (Life) according to the manufactures protocol. RNA from three replicates in each group was pooled together.
Label
Ambion WT Expression Kit (Applied Biosystems)
Label protocol
250ng of total RNA per group was amplified and labeled using the Ambion WT Expression Kit (Applied Biosystems) according to manufactures instructions.
Hybridization protocol
Standard protocol, Affymetrix Core center, Rigshospitalet, Copenhagen, Denmark
Scan protocol
Standard protocol, Affymetrix Core center, Rigshospitalet, Copenhagen, Denmark
