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Tumor-conditioned medium (CM) preparation

CT Chisaho Torii
NM Nako Maishi
TK Taisuke Kawamoto
MM Masahiro Morimoto
KA Kosuke Akiyama
YY Yusuke Yoshioka
TM Takashi Minami
TT Takuya Tsumita
MA Mohammad Towfik Alam
TO Takahiro Ochiya
YH Yasuhiro Hida
KH Kyoko Hida
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The tumor CM was collected from the culture supernatant of the tumor cells (A375SM or A375 cells) as previously described18. Briefly, the tumor cells (1 × 106 cells) were cultured in 10% FBS MEM for 24 h. The tumor CM was obtained from culture supernatant after passing through a 0.22 μm syringe filter (Millipore, Billerica, MA, USA). The control CM was collected from the culture supernatant from HMVECs in 10% FBS MEM in the same way as tumor CM. The HMVECs were treated with tumor CM or control CM in several periods in each experiment. When the cells were treated for 5 days, the medium was changed to freshly collected CM at the third day. The IL-6 level in each CM was measured using an IL-6 ELISA kit (R&D systems, Q6000B, Minneapolis, MN).

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