4.4. Cell Migration and Invasion Assays

Transwell assay was used to evaluate the migration and invasion abilities of the prostate cancer cells. Twenty-four-well Transwell chambers with an 8 µm pore size polycarbonate membrane (Corning Inc., Corning, NY, USA) were used in these assays. For the Transwell assays, the PLZF expression vector and PLZF siRNA were transfected into DU145 or LNCaP cells and resuspended at a concentration of 5 × 10⁴ cells in the top well with 200 µL serum-free RPMI 1640 medium. The lower chamber was filled with a 700 µL mixture of fresh and CMs (1:1) with 10% fetal bovine serum or neutralizing antibody CCL3 (R&D System, Minneapolis, MN, USA) were added as a chemoattractant. For cell migration assay, DU145 or LNCaP cells in a serum-free medium were seeded into the top chamber and incubated at 37 °C for 24 h. For cell invasion assay, the membrane was coated with Matrigel (Corning Inc., Corning, NY, USA) and incubated at 37 °C for 36 h. Cells on the top surface of the interface membrane were removed using a cotton swab. Migrated and invaded cells on the lower surface of the membrane were fixed with 10% formaldehyde, stained with a hematoxylin and eosin kit (Sysmex Corporation, Kobe, Japan), and counted under an optical microscope (100×, Nikon, Tokyo, Japan) from four random fields using ImageJ software (National Institutes of Health, Bethesda, MD, USA).