Concanavalin A Protocol for Felid Lymphocyte Culture and Chromosome Preparation

Chromosome preparation from peripheral blood in felids is often difficult because lymphocytes respond inconsistently to standard mitogens like phytohemagglutinin (PHA), resulting in low and variable mitotic indices. Here, we present a standardized and reproducible protocol for lymphocyte culture and metaphase chromosome preparation in felids using concanavalin A (ConA) as a mitogenic stimulus. A total of 300 µL of peripheral blood consistently produced optimal chromosome preparations and was cultured in RPMI 1640 medium supplemented with fetal bovine serum, antibiotics, and Concanavalin A (40 µg/mL), then incubated at 37 °C for 70 h. Mitotic arrest was induced with colchicine, and cells were subjected to hypotonic treatment and fixation using methanol–acetic acid before slide preparation and Giemsa staining. This approach consistently yielded high-quality metaphase spreads across the eight felid species examined, with mean values ranging from 32 ± 5 to 55 ± 7 metaphases per slide, while maintaining chromosome morphology appropriate for cytogenetic investigation. Compared with fibroblast-based approaches, this methodology is less invasive and more feasible for clinical and conservation applications because it relies on peripheral blood collection rather than tissue biopsy, reducing the invasiveness of sampling procedures. This optimized lymphocyte culture workflow enhances mitotic yield and repeatability, serving as a significant resource for cytogenetic research, particularly in veterinary genetics, evolutionary biology, and the conservation of endangered felid species.