Rabbit Immune Cell Function: In Vitro Assays for Immunological Assessment Studies Using Flow Cytometry

Repeated blood collection in long-term studies in rodents is challenging and may compromise animal welfare. Rabbits represent a robust alternative model for longitudinal studies due to their larger blood volume and ease of repeated sampling. However, standardized assays to assess rabbit immune function remain scarce. This study presents standardized and optimized flow cytometry–based protocols for evaluating oxidative burst, phagocytosis, and lymphocyte proliferation in rabbits. Oxidative burst and phagocytic activity were analyzed in heparinized whole blood using DCFH and fluorescently labeled Staphylococcus aureus. Lymphocyte proliferation was assessed in CFSE-labeled PBMC stimulated with ConA. Flow cytometric analysis enabled simultaneous quantification of reactive oxygen species (ROS) generation, phagocytic uptake, and CFSE dilution modeling. Following stimulation with S. aureus, rabbit heterophils exhibited ROS production in a median of 90.4% (IQR: 84.8–92.9%) of gated cells, with a median phagocytic uptake of 23.6% (IQR: 13.4–27.2%). PMA-stimulated cells showed near-complete oxidative burst (median 99.1%, IQR: 98.7–99.5%), confirming their functional similarity to mammalian neutrophils. Lymphocytes exhibited measurable proliferative responses to ConA, validating PBMC-based assays for adaptive immune assessment. These standardized methods offer a framework for investigating innate and adaptive immune functions in rabbits, contributing to immunotoxicological and safety evaluation studies.