Bridging Immunity and Hemostasis: Technical Foundations of Platelet-Leukocyte Co-cultures.

Platelets are immune cells with a great potential for the discovery of novel mechanisms regulating both innate and adaptive responses. Although it is known that the interaction between platelets and leukocytes plays a critical role in inflammation and thrombosis, the vast majority of experimental models focus on monotypic cell systems, ignoring the constant association of these two cell types in circulation and in the tissues. Co-culture systems that recapitulate this interplay are essential for understanding cellular processes and for evaluating therapeutic interventions. However, the technical requirements for isolating and handling these cells with such distinct physical and functional properties pose significant challenges. Here I provide a detailed guide for the isolation, characterization, and co-culture of human and murine platelets with leukocytes, while describing systematic procedures for obtaining high-purity platelet suspensions with minimal activation. Parallel instructions for isolating and differentiating leukocytes from human blood or mouse bone marrow are also provided. In addition, I outline flow cytometry-based methods to assess platelet purity and activation status using constitutive and activation-inducible markers. This comprehensive guide is aimed at experienced operators and newcomers alike, offering practical tips and troubleshooting advice to ensure reproducibility and biological relevance. By standardizing critical steps in co-culture protocols, this resource seeks to support high-quality experimental design in immunology and hemostasis research.