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.