Centrifuge-less red blood cell lysis and immunostaining of whole blood for flow cytometry using DA-Cell washer and plate

Abstract Blood cells are prime indicators of immuno-surveillance, and the ease of blood sampling makes blood analysis a key interest for clinical and research applications. While current flow cytometry methods are high-throughput and provide fine resolution in the segregation of white blood cell (WBC) populations, WBC enrichment involving red blood cell (RBC) lysis are laborious and typically performed manually, contributing to experimental variability especially as blood cells are sensitive to physical and chemical stress. We describe RBC lysis and leukocyte immunostaining on a centrifuge-less platform DA-Cell™, using a novel wall-less plate and laminar flow washer. The DropArray (DA) plate consists of an array of 96 hydrophilic spots surrounded by hydrophobic surface, which functions as a virtual wall that separates each spot. The maximum volume of each spot can be increased to 300μL with an insert. During lysis, WBC settle to the surface of the spot, allowing the spent lysis buffer to be removed. After removal of the insert, the plate goes through a gentle 3–4min laminar-flow washing process in the DA-Cell washer, decreasing cell washing time by at least 50% while eliminating centrifugation that stresses cells and disrupts antibody binding. In studies comparing mouse whole blood lysis (1X RBC Lysis buffer, eBioscience) and antibody staining by conventional tube centrifuge and DA-Cell, DA-Cell achieved dramatically higher staining index and improved resolution of cell cluster by flow cytometry. CD45+ leukocyte recovery and viability was uncompromised compared to conventional tube centrifuge. In summary, DA-Cell system provides gentle, fast and convenient blood lysis, while improving data quality with superior antibody staining.