Isosulfan blue sentinel lymph node biopsy enables reliable lymph node harvest and multicolor flow cytometry in mice.

Accurate and reproducible collection of the draining lymph nodes (DLNs) is essential for studying immune responses in preclinical models, yet technical variability and the small size of murine lymph nodes make consistent harvest challenging, particularly across users with differing surgical experience. To address this limitation, we adapted the clinical technique of isosulfan blue sentinel lymph node biopsy (IB-SLNB) for use in mice. Despite its widespread application in surgical oncology, IB has not been systematically evaluated for compatibility with fluorescence-based immunologic assays. Here, we show that IB-SLNB enables rapid and reliable identification of axillary DLNs, improving reproducibility of lymph node harvest. Importantly, IB labeling does not interfere with multicolor flow cytometry or downstream colorimetric analyses, preserving analytic fidelity. This facile, low-cost method enhances consistency in lymphatic tissue collection and supports high-quality immune profiling across diverse experimental contexts, including models of infection, cancer, and transplantation.