The mysterious case of missing lymphocytes: a cautionary tale of interinstitutional variability in outcomes of lung dissociation protocols

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 interinstitutional 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 merely 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 interinstitutional variability in lymphocyte outcomes remains unknown, this report serves as a cautionary tale against directly adopting lung dissociation protocols across institutions without reoptimization, and calls for careful inspection of cross-institutional reproducibility of previously described protocols.<b>NEW & NOTEWORTHY</b> 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, the authors share their experience with interinstitutional variability that led to the discovery that a protocol optimized for murine lung dissociation in one institution failed to reproduce similar lymphocyte outcomes elsewhere. This report, thus, serves as a cautionary tale against directly adopting tissue dissociation protocols across institutions without reoptimization.