Dual‐labelled antibodies for flow and mass cytometry: A new tool for cross‐platform comparison and enrichment of target cells for mass cytometry

Abstract

Antibody conjugates applicable in both conventional flow and mass cytometry would offer interesting options for cross-platform comparison, as well as the enrichment of rare target cells by conventional flow cytometry (FC) sorting prior to deep phenotyping by mass cytometry (MC). Here, we introduce a simple method to generate dual fluorochrome/metal-labelled antibodies by consecutive orthogonal labelling. First, we compared different fluorochrome-conjugated antibodies specific for CD4, such as FITC, Vio667, VioGreen or VioBlue for their compatibility with the conventional secondary MAXPAR labelling protocol. After labelling with Pr, the fluorescence emission spectra of all fluorochromes investigated retained their characteristics, and CD4 dual conjugates (DCs) provided consistent results in immune phenotyping assays performed by FC and MC. The phenotypical composition of CD4 T-cells was maintained after enrichment by FC sorting using different CD4 DCs. Finally, magnetic cell depletion was combined with FC sorting using CD19-VioBlue- Nd, CD20-VioGreen- Sm, CD27-Cy5- Er and CD38-Alexa488- Nd DC to enrich rare human plasmablasts to purities >80%, which allowed a subsequent deep phenotyping by MC. In conclusion, DCs have been successfully established for direct assay comparison between FC and MC, and help to minimise MC data acquisition time for deep phenotyping of rare cell subsets.

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

“…A lower throughput (<500 events per second) usually delivers data comprising fewer doublet events. Thus, in contrast to most fluorescence‐based flow cytometers with event acquisition rates of usually up to 10 000 events/s, acquisition times in mass cytometry are significantly longer and might necessitate pre‐enrichment of target cells prior to mass cytometric analysis . In addition, a CyTOF measurement recovers data for about 30–50% of the injected cells, while the remaining sample is lost, e.g., by accumulating on the walls of the spray chamber and injector.…”

Guidelines for the use of flow cytometry and cell sorting in immunological studies*

“…A lower throughput (<500 events per second) usually delivers data comprising fewer doublet events. Thus, in contrast to most fluorescence‐based flow cytometers with event acquisition rates of usually up to 10 000 events per second, acquisition times in mass cytometry are significantly longer and might necessitate pre‐enrichment of target cells prior to mass cytometric analysis . In addition, a CyTOF measurement recovers data for about 30–50% of the injected cells, while the remaining sample is lost, e.g.…”

Mass Cytometry for the Assessment of Immune Reconstitution After Hematopoietic Stem Cell Transplantation

“…This can impact the feasibility of detecting rare cell subsets in blood samples from HSCT patients during the first 2-weeks post-transplant, where the numbers of circulating immune cells may be very low and peripheral blood draw volume limited. In studies where particular mass cytometric analysis of rare populations is paramount, implementation of dual conjugated antibodies (metal and fluorescent tagged) can facilitate bead-free enrichment by fluorescent cell sorting prior to mass analysis ( 156 ). An additional limitation in mass cytometry is that samples are completely ablated by the plasma torch prior to acquisition, precluding the possibility of cell sorting and the recovery of viable cells for further assays.…”