Mass cytometers are time-of-flight (TOF) mass spectrometer-coupled flow cytometers (known as CyTOFs) that quantify the abundance of metal-tagged antibodies (Abs) or other cellular probes within single cell suspensions or laser-ablated tissue sections. While many strategies exist for covalently crosslinking to proteins, the Fluidigm MaxPar process is currently the most widely used and involves first loading a metal-chelating polymer with an elementally and isotopically enriched metal. Once the chelation sites have been filled, a maleimide moiety on the polymer is reacted with the free thiol groups on the partially reduced monoclonal immunoglobulin G (IgG) Ab to form an irreversible covalent bond. Here we describe modifications to the Fluidigm MaxPar protocol that increase the quantity of Ab per reaction up to 150 μg, introduce an initial Ab quality control step, utilize metal labels not included in the Fluidigm catalog, and provide an option to perform two reactions in one centrifugal filter.
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