No-lyse no-wash flow cytometry method for ex vivo study of human neutrophils

Neutrophils are vital immune cells involved in immunity via their effector roles and death. While their rapid response to the environment is crucial for immune function, it also makes them difficult to study. Here, we provide a no-lyse no-wash (NLNW) flow cytometry protocol to quantify neutrophil viability and death in whole blood with minimal sample handling. Viability of neutrophils was measured by Annexin V/7AAD staining in whole blood at 0 and 20 h. Associated cost, sample volume, and neutrophil survival were compared with traditional density-gradient isolated neutrophils. The NLNW analysis of whole blood immediately after collection showed ∼85% viable neutrophils. After 20 h of incubation, viability dropped to ∼70%, yet was significantly greater compared with isolated neutrophils, where half the population (∼44%) entered early-stage apoptosis. The NLNW flow cytometry method is an efficient and cost-effective method to measure neutrophil viability in small volumes of whole blood with minimal sample handling.