Matrigel precoated transwell chambers with 8 mm pores

Tumor cell invasion was measured by counting the number of tumor cells that invaded through matrigel pre-coated transwell chambers with 8-mm pores (BD Biosciences). On the top of inserts: 24 hour FBS starved; untreated; or 100 nM 24 hour triptolide treated tumor cells (KPC-1, Panc02 and S2-VP10, 1.5×10⁵ each) were placed. On the bottom chamber: untreated or 100 nM 24 hour triptolide treated CAFs (CAF-1, CAF-5 and SC00A5, 1.5×10⁵ each) in 1% FBS were added. The negative control group was added with 1% FBS in the bottom chamber. After incubation for 24 hours (KPC-1 and S2-VP10) to 48 hours (Panc02) for invasion, the invaded cells were fixed with 70% ethanol, stained with crystal violet, and 5 random fields were counted under a light microscope. Each experiment was repeated thrice.