Optimized membrane filtration for high-purity red blood cell isolation from human and murine samples

Multidisciplinary AbstractAccurate experimental analysis of red blood cells (RBC) warrants its purification from other blood components as even low-level contamination by unwanted cell populations can introduce substantial artifacts. Conventional purification methods, such as density-based separation followed by removal of intermediate layers, often leave significant residual contamination. Other approaches, including affinity-based separation or flow-based sorting, achieve high purity but are limited by low throughput, high cost, and impracticality for processing large sample volumes. Filtration-based strategies using cellulose matrices have been widely employed; however, these approaches are time-consuming, sensitive to variability in matrix preparation across laboratories, and constrained by fixed pore structures inherent to the material. We developed an RBC purification method based on commercially available filter membranes. This approach proved simple, cost-effective, and required no specialized equipment. Importantly, membrane pore size can be readily selected to match the physical properties of the RBC targeted population, improving purification efficiency and reproducibility. Using this membrane-based filtration protocol, we achieved effective removal of contaminating cells in human and murine RBC samples. Overall, this method provides a robust and inexpensive RBC purification strategy that supports reliable downstream functional and biochemical analyses across a wide range of experimental applications especially for low volume samples.