Surface Barcoding of Live PBMC for Multiplexed Mass Cytometry
Sample barcoding is a powerful method for harmonizing mass cytometry data. By assigning a unique combination of barcode labels to each cell sample, a set of individual samples can be pooled and further processed and acquired as a large, single sample. For assays that require uncompromised profiling of cell-surface markers on live cells, barcoding by metal-labeled antibodies targeting cell-surface epitopes is the barcoding approach of choice. Here we provide an optimized and validated protocol for cell-surface barcoding of ten PBMC samples with palladium-labeled β2-microglobulin (B2M) antibodies used in a 5-choose-2 barcoding scheme, for subsequent immune phenotyping by mass cytometry. We further provide details on the generation of palladium-labeled antibodies utilizing amine-reactive isothiocyanobenzyl-EDTA (ITCB-EDTA) that permits the implementation of antibody-based barcoding not interfering with lanthanide channels typically used for analyte detection in mass cytometry assays.
Key wordsMass cytometry CyTOF Cell-surface sample barcoding Antibody-based sample barcoding Palladium Immune monitoring β2-Microglobulin
The authors would like to thank all members of the DRFZ mass cytometry group, Michael D. Leipold, PhD, for discussing antibody labeling strategies, Dr. Lars Fransecky for providing leukemia samples, and Dr. Petra Henklein for providing access to the lyophilizer.
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