Lv150n

We used a non-inverted industrial fluorescence microscope (Nikon LV150N, Tokyo, Japan) with brightfield, darkfield, simple polarizing, double beam interferometry, and DIC attachments. To visualize fluorescence images, a 20× objective lens (NA/WD = 0.45/4.5 mm) was used, and a DS-Ri2 microscope CMOS camera (Nikon, 6fps [4908 × 3246 pixels] to 45 fps [1636 × 1088 pixels]) was used to capture the fluorescence images. Fluorescence excitation was provided by a 100 W mercury lamp. The excitation and emission of propidium iodide (PI) dye and calcein-AM were EX 535–550 nm/DM 575 nm/DM 580 nm and EX 470–540 nm/DM 505 nm/EM 510 nm.

THP-1 cells (1 × 10⁶) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Then THP-1 cells were differentiated to macrophages with 100 nmol/mL Phorbol-12-myristate 13-acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA) treatment for 48 h. An optical microscope (LV150N, Nikon, Japan) was used to verify cell differentiation by evaluating cell adhesion.
The macrophages (5 × 10⁵) were seeded into the upper well (0.4-μm pores; Corning, NY, USA) of 6-well plates, and the cancer cells were seeded into the lower wells of the 6-well plates. The ratio of macrophages and cancer cells was 1:2. Both groups of cells were allowed to adhere. Afterward, the upper wells and lower wells in the 6-well plates were replaced with fresh medium. Then, macrophages and cancer cells were co-cultured for 24 h. The migratory abilities of the co-cultured and mono-cultured cancer cells were determined using the transwell assay. The co-cultured macrophages (macrophage CO) and Kyse150 cells (Kyse150 CO) were cultured separately for an additional 24 hrs to investigate whether the cancer cells can self-stimulate. The resulting CM (Kyse150 CO CM and macrophage CO CM) was added to untreated Kyse150 cells.