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Animals randomly picked from all groups were terminated on day 30, and peripheral blood lymphocytes were extracted and co-incubated with the H22 tumor cells.
Lymphocytes were extracted from peripheral blood by layering onto Lymphoprep (Axis-Shield) and taking the interface.
The former two were mostly isolated from the peripheral blood of the mice, and lymphocytes were extracted from the spleen.
Lymphocytes were extracted from the EDTA tubes (total 13 ml whole blood) using Lymphoprep tubes (Nycomed Pharma AS, Oslo, Norway) and stored at -20°C.
Briefly, areas of interest (2 3 mm) were microdissected from paraffin-embedded formalin-fixed tissue sections and digested with proteinase K. MCF7 cell-line DNA and reference control DNAs from peripheral blood lymphocytes were extracted using a Progenome kit (Progen, Ipswich, Australia).
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DNA from peripheral lymphocytes was extracted using a DNA-extraction kit from Stratagene, according to the manufacturer's protocol.
Total RNA of 100 000 lymphocytes was extracted and complementary DNA synthesis was performed by standard procedures.
For PCR experiments, genomic DNA from peripheral blood lymphocytes was extracted as described [ 10].
Genomic DNA from lymphocytes was extracted using the QiaAmp Blood kit according to the manufacturer's instructions (Qiagen, Hilden, Germany).
DNA from whole blood lymphocytes was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) and was stored at −20 °C until use for genotyping.
The genomic DNA from peripheral blood lymphocytes was extracted by the QIAamp DNA Blood Maxi Kit (Qiagen, Hilden, Germany) for family ACC1 and by a salting-out procedure for family DS1 and DS2.
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