Preparation of Walker 256 rat mammary gland carcinoma cells

Female rats weighing 60–80 g were injected with 0.5 mL of 1 × 10⁷ cells/mL Walker 256 cells (Shanghai Jiuzhi Biotechnology Co. Ltd., Shanghai, China) into the abdominal cavity. After seven days, the ascites fluid was collected aseptically. The cells were centrifuged at 1200 r/min for 3 min, washed three times with 10 mL of sterile phosphate-buffered saline (PBS), counted using a hemocytometer, diluted to a concentration of 1 × 10⁶ cells/mL for inoculation, and preserved on ice prior to surgery. For the sham group, the same volume of PBS was prepared for injection.