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For preconcentration and cleanup, Oasis HLB (60 mg, 3 mL) and Thermo HyperSep Retain PEP (60 mg, 3 mL) SPE cartridges, each conditioned with 5 mL methanol and equilibrated with 5 mL Milli-Q water, were used.
Freezing behavior was analyzed for 20 s after the presentation of each conditioned tone.
Gastric cancer cells and fibroblasts were incubated in 3 ml DMEM containing 2% FCS with 50% each conditioned medium.
Each conditioned medium was centrifuged to remove debris (1500 rpm for 10 min at 4°C) before use.
Fifteen microliters of each conditioned medium and zymography sample buffer were combined and incubated at room temperature for 5 minutes.
During a training trial, the whole arena was illuminated with alternating green and blue light (60 s each; conditioned stimuli = CS), one of the colors was paired with reinforcement (unconditioned stimulus = US).
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Each condition used different groups of genes.
Each condition was proved every day.
Each condition lasted two weeks.
Each condition had a concurrent control group.
Each condition comprised 3 consecutive nights.
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