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Published on: January 22, 2021
FlowCLOc, a New Tool for Selecting the Most Appropriate Antibodies in Flow Cytometry
Valentina Serra1, Valeria Orrù1, Sandra Lai1
1Institute for Genetic and Biomedical Research, National Research Council, 08045 Lanusei, Italy.
Insights
Cryopreservation significantly alters immune cell marker expression in flow cytometry. A new public database, FlowCLOc, catalogs these changes to aid in selecting reliable antibodies for frozen and fresh samples.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Flow cytometry is crucial for immunophenotyping circulating immune cells.
- Cryopreservation of samples can impact cell recovery and marker expression, potentially affecting experimental results.
Purpose of the Study:
- To systematically evaluate the impact of cryopreservation on the expression of a wide range of cell surface markers.
- To develop a resource for optimizing flow cytometry panel design for both fresh and frozen samples.
Main Methods:
- Comparison of 438 antibodies on B, T, NK-enriched cells, and monocytes from fresh versus cryopreserved lympho-monocytes and blood.
- Quantitative assessment of marker expression changes exceeding 20%.
Main Results:
- Cryopreservation significantly altered the expression of 283 markers in lympho-monocytes and 262 in blood.
- A substantial proportion of measured markers showed >20% change post-cryopreservation.
Conclusions:
- Careful evaluation of antibody performance is essential when using frozen samples for flow cytometry.
- The FlowCLOc database provides a valuable tool to simplify panel design and improve data reliability for frozen and fresh samples.
Abstract:
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 time-consuming preliminary tests to select the most appropriate and specific markers for staining both frozen and fresh samples.
