Rigor and reproducibility are vital to scientific advancement. It is unclear whether a protocol optimized for tissue dissociation in one institution performs well universally. Here, we share our brand-new lab's experience with inter-institutional variability that led to the discovery that a protocol optimized for murine lung dissociation at Boston University (BU) fails to reproduce similar CD4+ T cell, CD8+ T cell, and B cell outcomes at the University of Michigan at Ann Arbor (U-M). We report that the type 2 collagenase-based protocol from BU yields reduced numbers of lung lymphocytes at U-M, and this appeared to be a result of harsher collagenase activity despite using identical protocols, reagents, and vendors at both institutions. This variability could not be explained by higher Ca2+ levels in Ann Arbor water (which we posited may heighten the collagenase activity) but instead appeared to be due to technical details within the protocol that led to the protocols behaving in an institution-specific manner. Indeed, we find that mere switching between the protocol from BU and a newly optimized protocol at U-M was sufficient to improve (or worsen) lymphocyte yields from murine lungs when synchronously performed at both institutions. Taken together, although the reason(s) for the inter-institutional variability in lymphocyte outcomes remains unknown, this report serves as a cautionary tale against directly adopting lung dissociation protocols across institutions without re-optimization, and calls for careful inspection of cross-institutional reproducibility of previously described protocols.
Keywords: Flowcytometry; Rigor and Reproducibility; collagenase; lung dissociation; lymphocytes.