A Universal Live Cell Barcoding-Platform for Multiplexed Human Single Cell Analysis.

Single-cell barcoding enables the combined processing and acquisition of multiple individual samples as one. This maximizes assay efficiency and eliminates technical variability in both sample preparation and analysis. Remaining challenges are the barcoding of live, unprocessed cells to increase downstream assay performance combined with the flexibility of the approach towards a broad range of cell types. To that end, we developed a novel antibody-based platform that allows the robust barcoding of live human cells for mass cytometry (CyTOF). By targeting both the MHC class I complex (beta-2-microglobulin) and a broadly expressed sodium-potassium ATPase-subunit (CD298) with platinum-conjugated antibodies, human immune cells, stem cells as well as tumor cells could be multiplexed in the same single-cell assay. In addition, we present a novel palladium-based covalent viability reagent compatible with this barcoding strategy. Altogether, this platform enables mass cytometry-based, live-cell barcoding across a multitude of human sample types and provides a scheme for multiplexed barcoding of human single-cell assays in general

GateFinder: projection-based gating strategy optimization for flow and mass cytometry

Motivation: High-parameter single-cell technologies can reveal novel cell populations of interest, but studying or validating these populations using lower-parameter methods remains challenging.Results: Here, we present GateFinder, an algorithm that enriches high-dimensional cell types with simple, stepwise polygon gates requiring only two markers at a time. A series of case studies of complex cell types illustrates how simplified enrichment strategies can enable more efficient assays, reveal novel biomarkers and clarify underlying biology

A Universal Live Cell Barcoding-Platform for Multiplexed Human Single Cell Analysis.

Single-cell barcoding enables the combined processing and acquisition of multiple individual samples as one. This maximizes assay efficiency and eliminates technical variability in both sample preparation and analysis. Remaining challenges are the barcoding of live, unprocessed cells to increase downstream assay performance combined with the flexibility of the approach towards a broad range of cell types. To that end, we developed a novel antibody-based platform that allows the robust barcoding of live human cells for mass cytometry (CyTOF). By targeting both the MHC class I complex (beta-2-microglobulin) and a broadly expressed sodium-potassium ATPase-subunit (CD298) with platinum-conjugated antibodies, human immune cells, stem cells as well as tumor cells could be multiplexed in the same single-cell assay. In addition, we present a novel palladium-based covalent viability reagent compatible with this barcoding strategy. Altogether, this platform enables mass cytometry-based, live-cell barcoding across a multitude of human sample types and provides a scheme for multiplexed barcoding of human single-cell assays in general

A platinum-based covalent viability reagent for single-cell mass cytometry

In fluorescence-based flow cytometry, cellular viability is determined with membrane-impermeable fluorescent reagents that specifically enter and label plasma membrane-compromised nonviable cells. A recent technological advance in flow cytometry uses antibodies conjugated to elemental metal isotopes, rather than to fluorophores, to allow signal detection by atomic mass spectrometry. Unhampered by the limitations of overlapping emission fluorescence, mass cytometry increases the number of parameters that can be measured in single cells. However, mass cytometry is unable to take advantage of current fluorescent viability dyes. An alternative methodology was therefore developed here in which the platinum-containing chemotherapy drug cisplatin was used to resolve live and dead cells by mass cytometry. In a 1-min incubation step, cisplatin preferentially labeled nonviable cells from both adherent and suspension cultures, resulting in a platinum signal quantifiable by mass cytometry. This protocol was compatible with established sample processing steps for intracellular cytometry. Furthermore, the live/dead ratios were comparable between mass- and fluorescence-based cytometry. Importantly, although cisplatin is a known DNA-damaging agent, a 1-min "pulse" of cisplatin did not induce observable DNA damage or apoptotic responses even within 6-h post-exposure. Cisplatin can therefore be used as a viability reagent for a wide range of mass cytometry protocols

Scalable Conjugation and Characterization of Immunoglobulins with Stable Mass Isotope Reporters for Single-Cell Mass Cytometry Analysis

The advent of mass cytometry (CyTOF®) has permitted simultaneous detection of more than 40 antibody parameters at the single-cell level, although a limited number of metal-labeled antibodies are commercially available. Here we present optimized and scalable protocols for conjugation of lanthanide as well as bismuth ions to immunoglobulin (Ig) using a maleimide-functionalized chelating polymer and for characterization of the conjugate. The maleimide functional group is reactive with cysteine sulfhydryl groups generated through partial reduction of the Ig Fc region. Incubation of Ig with polymer pre-loaded with lanthanide ions produces metal-labeled Ig without disrupting antigen specificity. Antibody recovery rates can be determined by UV spectrophotometry and frequently exceeds 60%. Each custom-conjugated antibody is validated using positive and negative cellular control populations and is titrated for optimal staining at concentrations ranging from 0.1 to 10 μg/mL. The preparation of metal-labeled antibodies can be completed in 4.5 h, and titration requires an additional 3-5 h

Drugging MYCN through an Allosteric Transition in Aurora Kinase A

MYC proteins are major drivers of cancer yet are considered undruggable because their DNA binding domains are composed of two extended alpha helices with no apparent surfaces for small-molecule binding. Proteolytic degradation of MYCN protein is regulated in part by a kinase-independent function of Aurora A. We describe a class of inhibitors that disrupts the native conformation of Aurora A and drives the degradation of MYCN protein across MYCN-driven cancers. Comparison of cocrystal structures with structure-activity relationships across multiple inhibitors and chemotypes, coupled with mechanistic studies and biochemical assays, delineates an Aurora A conformation-specific effect on proteolytic degradation of MYCN, rather than simple nanomolar-level inhibition of Aurora A kinase activity

Alternate mechanisms of initial pattern recognition drive differential immune responses to related poxviruses

Vaccinia immunization was pivotal to successful smallpox eradication. However, the early immune responses that distinguish poxvirus immunization from pathogenic infection remain unknown. To address this, we developed a strategy to map the activation of key signaling networks in vivo and applied this approach to define and compare the earliest signaling events elicited by immunizing (vaccinia) and lethal (ectromelia) poxvirus infections in mice. Vaccinia induced rapid TLR2-dependent responses, leading to IL-6 production, which then initiated STAT3 signaling in dendritic and T cells. In contrast, ectromelia did not induce TLR2 activation, and profound mouse strain-dependent responses were observed. In resistant C57BL/6 mice, the STAT1 and STAT3 pathways were rapidly activated, whereas in susceptible BALB/c mice, IL-6-dependent STAT3 activation did not occur. These data link early immune signaling events to infection outcome and suggest that activation of different pattern-recognition receptors early after infection may be important in determining vaccine efficacy