Standardizing murine lamina propria leukocyte isolation: Peeling back the layers of intestinal immunology for...

The intestinal lamina propria harbors a diverse repertoire of innate and adaptive immune cell populations, whose dynamic regulation is critical for maintaining mucosal immune homeostasis and for mounting effective inflammatory responses to infection or tissue injury. Accurate characterization of these populations in murine models requires reliable isolation of viable cells while preserving surface marker integrity. However, the heterogeneous cellular composition of the lamina propria poses significant technical challenges, and variability in isolation protocols has contributed to reproducibility issues across studies in mouse experimental models. In particular, enzymatic digestion can markedly affect cell viability and surface molecule expression, with outcomes depending on the choice of enzyme and the inclusion of additives such as deoxyribonuclease I (DNase I) and/or dithiothreitol (DTT). Standardization of isolation methods in the immunology field is therefore essential to ensure consistent recovery of viable immune cells suitable for downstream analyses. To promote reproducibility across laboratories, we introduce a minimal yet comprehensive reporting checklist, together with a systematic overview of the key experimental variables that govern the yield, viability, and phenotypic integrity of lamina propria leukocytes (LPLs) in mice.