FlowCLOc, a New Tool for Selecting the Most Appropriate Antibodies in Flow Cytometry

Circulating immune cells are frequently phenotyped by flow cytometry starting from frozen samples. However, cryopreservation can affect marker expression and cell recovery. To understand which antigens are detectable and reliable after sample cryopreservation, we compared 438 antibodies measured on B, T, and NK-enriched cells and monocytes in frozen lympho-monocytes and blood with the corresponding fresh blood samples. Cryopreservation affected the expression of 283 markers in lympho-monocytes and 262 in blood, modifying them by more than 20% with respect to fresh blood. Thus, it is essential to carefully evaluate antibody performance when working with frozen samples. To maximize the usability of our results, make them publicly accessible and ready to visualize, we created a catalogue of marker expression variability before and after freezing, namely FlowCLOc. This catalogue simplifies flow cytometry panel design, reducing timeconsuming preliminary tests to select the most appropriate and specific markers for staining both frozen and fresh samples.