Culturing Primary DRG Neurons with Breast Cancer Cells

Primary cultures of DRG were established according to a published protocol [21 (link)]. Two days old Sprague Dawley rats were obtained from the Experimental Animal Center of Dalian medical university (permit number SCXK 2008-0002). Briefly, DRG was dissected from newborn Sprague Dawley (2 days old) and pooled in an L-15 medium (Gibco) on ice. The DRG was then transferred to the lower chamber of the Transwell which was covered with rat tail collagen Type I (Invitrogen, USA). After 24 h, the DRG neurons were purified with 5 μg/ml cytarabine (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). MDA-MB-231 cells were cultured in the upper chambers of the 6-well plates at a density of 1 × 10⁴ cells/cm². The control group contained only the DRG (referred to as the DRG group). The cells were cultured at 37°C in a 5% CO₂ incubator for 7 days and the medium was changed every 2 days. Using NE, NGF, and β₂-AR/TrkA blocker (Shandong Topscience, Rizhao, China) pretreatment, the axon growth of each group of DRG neurons was detected by immunofluorescence staining.