Stabilizing Antibody Cocktails for Mass Cytometry

Abstract

Mass cytometry is increasingly employed in larger immune profiling studies involving data acquisitions across several days and multiple sites. For gaining a maximum of information from respective data by computational analyses, several techniques have been developed to minimize noise in mass cytometric data sets, such as sample banking, standardized instrument setup, sample barcoding, and signal normalization. However, the repeated preparation of cocktails composed of isotope-tagged antibodies remained a significant source of error. We here show that premixed antibody cocktails fail to deliver expected staining patterns when stored at 4 C for 4 weeks. As a solution, we developed and tested a cryopreservation method for highly multiplexed antibody cocktails for mass cytometry including lanthanide, palladium, and platinum conjugates that yielded stable staining patterns for at least 9 months when stored at temperatures below −80 C. Using frozen aliquots of antibody cocktails is an economic and flexible approach to significantly improve data consistency in large mass cytometry studies with repetitive staining/measurement cycles spanning several days or involving multiple data acquisition sites.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

“…Generally, the implementation of standardized tuning, sample barcoding (described in greater detail in Chapter VIII Section 2 Barcoding in cytometric assays) , signal normalization according to bead standards , and spillover compensation , and Ab cocktail cryopreservation secure the generation of high quality data in mass cytometry.…”

Reproducibility of Flow Cytometry Through Standardization: Opportunities and Challenges

“…Thus, the availability of stable reagent mixes on the market that contain an entire panel or allow at least a building block approach to a panel design (backbone reagents in the mix and few reagents added individually) would help to reduce the design and performance variability. This benefit is documented by some of the newer technologies, for example, mass cytometry (84). Reagent sets can be supplied in blocs, thus allowing analysis tools to be tailored to a given panel (85) and thus allow for direct comparability of the resulting immunophenotype patterns between projects and between laboratories.…”

Cyt‐Geist: Current and Future Challenges in Cytometry: Reports of the CYTO 2019 Conference Workshops

“…To track and minimize technical variation with antibody staining, facilitators have developed lyophilized antibody panels to minimize variability in preparing cocktails and thus streamlining the sample staining workflow. Additionally, there was also a discussion regarding preparing a master antibody cocktail mix that can be aliquoted and stored at –80°C and thawed when needed .…”