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

Area of Science:

• Immunology
• Flow Cytometry
• Animal Models

Background:

• Rodent models present challenges for longitudinal studies due to repeated blood collection impacting animal welfare.
• Rabbits offer a viable alternative for longitudinal studies owing to larger blood volumes and easier sampling.
• Standardized assays for assessing rabbit immune function are currently limited.

Purpose of the Study:

• To establish and optimize standardized flow cytometry protocols for evaluating key immune functions in rabbits.
• To assess oxidative burst, phagocytosis, and lymphocyte proliferation in rabbits using flow cytometry.
• To provide a framework for immunotoxicological and safety evaluation studies in rabbits.

Main Methods:

• Oxidative burst and phagocytosis were analyzed in whole blood using DCFH and fluorescently labeled *Staphylococcus aureus*.
• Lymphocyte proliferation was assessed in peripheral blood mononuclear cells (PBMCs) labeled with CFSE and stimulated with ConA.
• Flow cytometry was utilized for simultaneous quantification of reactive oxygen species (ROS) generation, phagocytic uptake, and CFSE dilution.

Main Results:

• Rabbit heterophils demonstrated significant ROS production (median 90.4%) and phagocytic activity (median 23.6%) upon *S. aureus* stimulation.
• Phagocyte oxidative burst reached near-complete levels (median 99.1%) with PMA stimulation, aligning with mammalian neutrophils.
• CFSE-based assays confirmed measurable lymphocyte proliferative responses to ConA, validating adaptive immune assessment.

Conclusions:

• Standardized flow cytometry protocols provide a reliable method for assessing innate and adaptive immune responses in rabbits.
• These validated assays support the use of rabbits as a model for immunotoxicological and safety evaluations.
• The developed protocols contribute to advancing the use of rabbits in preclinical research and comparative immunology.