Proliferation Assay Using Cryopreserved Porcine Peripheral Mononuclear Cells Stimulated With Concanavalin A and Analyzed With FCS Express<sup>TM</sup> 7.18 Software

In vitro lymphocyte proliferation assays are essential for assessing immune responses and antiproliferative drug efficacy. Such assays rely on antigen presentation or mitogen stimulation, with performance determined by reagent concentration and incubation time. Although splenocytes are often used, peripheral blood mononuclear cells (PBMCs) offer more accessible and practical sampling. However, a streamlined protocol for porcine PBMCs proliferation with robust batch analysis has been lacking. We therefore developed a detailed workflow for inducing proliferation in cryopreserved porcine PBMCs using 5 μg/mL concanavalin A (ConA). The protocol covers cell isolation, cryopreservation, ConA stimulation, CD4+ T-cell staining, flow cytometry acquisition and gating on an Attune NxT instrument, and batch analysis with FCS Express^TM 7.18. This approach yielded 78.9% viable cells, of which 33.8% were CD4+ lymphocytes. Moreover, 93.9% (n = 216) of cells proliferated, yielding up to nine cell generations. Batch analysis in FCS Express^TM enhanced the accuracy and interpretation of proliferation metrics. This validated protocol provides a reliable framework for generating consistent proliferation data in porcine immunology studies. Key features • Optimized proliferation assay for cryopreserved porcine PBMCs: Ideal for immunological studies and biomedical research using swine models. • Precise acquisition and gating with Attune NxT flow cytometer: Ensures accurate identification and analysis of CD4+ T lymphocytes. • Robust data analysis using FCS Express^TM 7.18 software: Facilitates reliable interpretation of lymphocyte proliferation and division indexes. • Efficient 4.5-day protocol: Enables timely and reproducible experiments in clinical and research settings involving porcine immune cells. Graphical overview Proliferation assay process from cryopreserved porcine peripheral blood mononuclear cells (PBMCs).