Diff stain set

The chemicals including berberine and paclitaxel were offered by Meilunbio. The purity of berberine was more than 99.8%. 2-Deoxy-D-glucose (2-DG) was purchased from Meck. The apoptosis detection kit (Annexin V-PI Staining) was purchased from Keygen Biotech (Nanjing, China). The Diff-Stain Set was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

After 12 days of adipocyte induction, the cells were cocultured with MDA-MB-231 and MDA-MB-468 human breast cancer cells using a Transwell culture system (0.4 μm pore size, Corning). 3.0 × 10⁵ MDA-MB-231 and MDA-MB-468 cells were seeded in the top chamber. Breast cancer cells were cocultured with adipocytes for 3 h, 6 h, 12 h, 24 h, 48 h and 72 h. MDA-MB-231 and MDA-MB-468 cells were cultured alone as controls and were evaluated at the same time points. To evaluate the effects of adipocyte coculture on breast cancer tumor cell migration, after 3 days of coculture with MDA-MB-231 and MDA-MB-468 cells, tumor cells were used to conduct migration assays using 24-well Boyden chambers containing inserts (8 μm, pores, BD Biosciences, USA). 5 × 10⁴ cells were cultured in the upper wells of Transwell chambers and allowed to migrate toward 10% FBS in the bottom wells. Similar migration assays were conducted with monocultured tumor cells. After 12 h, migrated cells were stained with Diff-Stain Set (Jiancheng), photographed, and counted using ImageJ software.