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Mass cytometry permits the high dimensional analysis of cellular systems at single-cell resolution with high throughput in various areas of biomedical research. Here, we provide a state-of-the-art protocol for the analysis of human peripheral blood mononuclear cells (PBMC) by mass cytometry. We focus on the implementation of measures promoting the harmonization of large and complex studies to aid robustness and reproducibility of immune phenotyping data., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers., (© 2021 Wiley-VCH GmbH.)

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mass cytometry is increasingly employed in larger immune profiling studies involving data acquisitions across several days and multiple sites. For gaining a maximum of information from respective data by computational analyses, several techniques have been developed to minimize noise in mass cytometric data sets, such as sample banking, standardized instrument setup, sample barcoding, and signal normalization. However, the repeated preparation of cocktails composed of isotope-tagged antibodies remained a significant source of error. We here show that premixed antibody cocktails fail to deliver expected staining patterns when stored at 4°C for 4 weeks. As a solution, we developed and tested a cryopreservation method for highly multiplexed antibody cocktails for mass cytometry including lanthanide, palladium, and platinum conjugates that yielded stable staining patterns for at least 9 months when stored at temperatures below -80°C. Using frozen aliquots of antibody cocktails is an economic and flexible approach to significantly improve data consistency in large mass cytometry studies with repetitive staining/measurement cycles spanning several days or involving multiple data acquisition sites. © 2019 International Society for Advancement of Cytometry., (© 2019 International Society for Advancement of Cytometry.)

Polystyrene beads are broadly applied in flow cytometry. Implementing bead-based assays in mass cytometry is desired but hampered by the lack of an elemental label required for their detection. In this study, we introduce stable osmium tetroxide labeling as a universal approach for generating functionalized beads readily detectable by mass cytometry. We demonstrate the utility of osmium-labeled beads for signal spillover compensation in mass cytometry, and, strikingly, their application in quantitative Ab-binding capacity assays combined with high-dimensional profiling of human PBMC enabled the systematic assessment of receptor expression profiles across large numbers of cellular phenotypes. This analysis confirmed increased monocytic Siglec-1 expression in active systemic lupus erythematosus patients and, additionally, revealed interrelated reductions of CD4 expression by regulatory and memory CD4 T cells and HLA-DR expression by myeloid dendritic cells, pointing toward defective cross-talk at the immunological synapse that may limit immune responses in systemic lupus erythematosus. By converting conventional flow cytometry beads into beads suitable for mass cytometry, our approach paves the way toward the broad implementation of bead-based assays in high-dimensional cell profiling studies by mass cytometry in biomedical research., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Receptor occupancy, the ratio between amount of drug bound and amount of total receptor on single cells, is a biomarker for treatment response to therapeutic monoclonal antibodies. Receptor occupancy is traditionally measured by flow cytometry. However, spectral overlap in flow cytometry limits the number of markers that can be measured simultaneously. This restricts receptor occupancy assays to the analysis of major cell types, although rare cell populations are of potential therapeutic relevance. We therefore developed a receptor occupancy assay suitable for mass cytometry. Measuring more markers than currently available in flow cytometry allows simultaneous receptor occupancy assessment and high-parameter immune phenotyping in whole blood, which should yield new insights into disease activity and therapeutic effects. However, varying sensitivity across the mass cytometer detection range may lead to misinterpretation of the receptor occupancy when drug and receptor are detected in different channels. In this report, we describe a method for optimization of mass cytometry receptor occupancy measurements by using antibody-binding quantum simply cellular (QSC) beads for standardization across channels with different sensitivities. We evaluated the method in a mass cytometry-based receptor occupancy assay for natalizumab, a therapeutic antibody used in multiple sclerosis treatment that binds to α4-integrin, which is expressed on leukocyte cell surfaces. Peripheral blood leukocytes from a treated patient were stained with a panel containing metal-conjugated antibodies for detection of natalizumab and α4-integrin. QSC beads with known antibody binding capacity were stained with the same metal-conjugated antibodies and were used to standardize the signal intensity in the leukocyte sample before calculating receptor occupancy. We found that QSC bead standardization across channels corrected for sensitivity differences for detection of drug and receptor and generated more accurate results than observed without standardization. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry., (© 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of 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.

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 141 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- 142 Nd, CD20-VioGreen- 147 Sm, CD27-Cy5- 167 Er and CD38-Alexa488- 143 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., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mass cytometry has pioneered >40-parameter single-cell analyses that allow for the characterization of complex cellular networks at unprecedented depth. Up to 135 parameters can be simultaneously detected, but limited availability of metal tags suitable for labeling of specific probes prevents optimal exploitation of the analytical capacity of mass cytometers. To this end, we here establish the application of elemental silver nanoparticles (AgNP) of different size for reporting cell surface antigens on human leukocytes in mass cytometry assays. The mass channels at 107 Da and 109 Da are uniquely occupied by silver isotopes and do not interfere with other mass cytometry reagents. Streptavidin-coated AgNP (SA-AgNP) facilitated distinct and specific detection of various antigens, such as CD8, CD244 and CD294 on peripheral blood leukocytes pre-incubated with respective biotinylated primary antibodies. Signal intensities elicited by 40 nm-sized AgNP allowed specific detection of the low abundance antigen CD25 on both, peripheral blood regulatory T cells and CD25 lo CD127 + CD4 + T cells, enabling their distinct clustering in viSNE plots. SA-AgNP were of high elemental purity, showed minor background binding to cells in immunoassays, and were compatible with previously established staining protocols for PBMC and leukocytes, facilitating their use in complex mass cytometry panels. Considering the synthesis of AgNP from isotopically purified silver, the usage of AgNP extends the analytical capacity of mass cytometry panels by one, prospectively two, additional parameters, suitable for the detection of cellular targets of low abundance. © 2016 International Society for Advancement of Cytometry., (© 2016 International Society for Advancement of Cytometry.)

Mass cytometry is developing as a means of multiparametric single-cell analysis. In this study, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a cytometry by time of flight instrument. Using six different anti-CD45 Ab conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and it reduces wet work and Ab consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45 barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and it should be applicable to fluorescence flow cytometry as well., (Copyright © 2015 by The American Association of Immunologists, Inc.)