Standardized human granulocyte isolation using Mono-Poly™ resolving medium: protocol development, validation, and comparison with Lymphoprep™

High purity neutrophil preparations are required for studying neutrophil biology and function. Although density gradient centrifugation is the most commonly used isolation technique, the choice of gradient medium can significantly affect cell purity and downstream applications. The stepwise method of using Mono-Poly™ Resolving Medium (M-PRM) for human peripheral blood granulocyte isolation is presented here alongside the Lymphoprep™ protocol, as these are the two commercially available density-gradient media selected for comparison in this study. Both protocols were optimized and validated by flow cytometric analysis using sequential FSC/SSC gating, singlet discrimination, and CD15-positive granulocyte phenotype identification. The use of M-PRM resulted in significantly higher average granulocyte purity (95.3%) than Lymphoprep™ (73.9%). This comparative protocol provides a practical guide for selecting an appropriate density gradient medium based on experimental requirements and offers a standardized workflow for obtaining granulocyte-enriched cell preparations suitable for downstream immunological and functional studies.