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Conclusion:
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Re-using a compensation matrix over a 6½-months period yielded results comparable to the Gold Standard (new compensation each day) when using fixed PMT voltages over time. In contrast, using BD Application Settings (adjusted PMTs) resulted in larger day-to-day variations. This larger variation seemed, at least in part, to be a result of “over-adjustment” of PMTs by Application Settings. Thus, the present findings call for caution in using Application Settings to improve stability of cell subset quantifications and especially MFI measurements in longitudinal studies. This is of special importance when also re-using a compensation matrix. As an alternative, we recommend inclusion of reference particles (e.g. Rainbow beads) to confirm cytometer stability. With a verified stable cytometer, and using fixed PMT settings, for some experiments re-use of a compensation matrix over time may be feasible. However, we do encourage special caution with this approach, and emphasize the opinion by most flow cytometry experts, that running fresh compensation samples with each experiment is the best practice. We encourage flow cytometry researchers from other institutions to reproduce these experiments.
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