Optimization of Erythrocyte Lysis Protocols for Robust and Reproducible T Cell Analysis in Murine Peripheral Blood, Spleen, Lung, and Bone Marrow

Flow cytometry analysis of T lymphocytes in murine peripheral blood, spleen, lung, and bone marrow is critical for immune studies. However, inconsistent erythrocyte lysis remains a major challenge-incomplete lysis obscures lymphocyte detection, while excessive lysis damages T cells. Moreover, the variable performance of commercial lysis buffers across tissues and protocols complicates reproducibility. To address these challenges, we first established a lysis buffer that provides a relatively balanced effect on erythrocyte lysis and lymphocyte protection by systematically comparing BD, BioLegend, and Biosharp buffers in murine peripheral blood. Given the high content of red blood cells (RBCs) in peripheral blood, we used it as a screening model to identify a buffer that effectively lysed RBCs while maintaining lymphocyte integrity. The BD lysing solution demonstrated superior performance, achieving effective erythrocyte removal with 41.40% CD3+ cell of the lymphocyte population at 1-min lysis (compared to BioLegend: 38.70% and Biosharp: 36.90% at their recommended durations). This buffer was then used as the unified buffer for subsequent multitissue optimization, ensuring a balanced effect across different tissue types. Building on this foundation, we developed a standardized framework, which resolves tissue-specific barriers. Spleen: Balanced erythrocyte clearance was achieved using 150 μL/million cells, yielding 67.23% ± 8.21% CD3+ lymphocytes. CD4+ and CD8+ T cell subsets constituted 58.50% ± 0.85% and 36.63% ± 0.85% of CD3+ cells, respectively (CD4/CD8 ratio: 1.60 ± 0.60). Lung: Adaptive processing volumes (30-150 μL/million cells) accommodated variable dissociation yields (1-5 × 106 cells/sample). CD45+ cells represented 42.87% ± 6.05% of live cells, with CD3+ cells dominating the lymphocyte compartment (96.57% ± 1.35%). CD4+ (58.50% ± 1.44%) and CD8+ (39.23 ± 1.12%) T cell proportions mirrored splenic distribution. Bone marrow: Erythrocyte depletion at 33.3 μL/million cells preserved rare lymphocytes (6.53% ± 0.30% of total cells). CD3+ cells comprised 21.80% ± 3.26% of CD45+ lymphocytes, exhibiting distinct subset ratios (CD4+: 57.34% ± 4.60%; CD8+: 50.50% ± 2.78%). Collectively, these protocols enable robust, operator-independent T cell analysis across murine immune compartments, providing a universal standard for translational immunology studies.