Optimizing the Structure of a Pt Metal-Chelating Polymer to Reduce Nonspecific Binding for Mass Cytometry.

Mass cytometry is a bioanalytic tool based on atomic mass spectrometry for detecting biomarker expression on individual cells. Current reagents employ metal-chelating polymers binding isotopes of hard metal ions. Polymers bearing chelators for soft metal ions offer the promise for a large increase in multiplexing capabilities, but examples reported so far often have unacceptably high levels of nonspecific binding (NSB).

Testing a Nanoparticle Reagent for Imaging Mass Cytometry.

Mass cytometry (MC), a powerful single-cell analysis technique, has limitations in detecting low-abundance biomarkers. Nanoparticle (NP) reagents offer the potential for enhancing sensitivity by carrying large numbers of heavy metal isotopes. Here, we report NP reporters for imaging mass cytometry (IMC) based on NaYF:Yb/Er NPs.

Reagents for Mass Cytometry.

Mass cytometry (cytometry by time-of-flight detection [CyTOF]) is a bioanalytical technique that enables the identification and quantification of diverse features of cellular systems with single-cell resolution. In suspension mass cytometry, cells are stained with stable heavy-atom isotope-tagged reagents, and then the cells are nebulized into an inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS) instrument. In imaging mass cytometry, a pulsed laser is used to ablate ca.

Polymeric dipicolylamine based mass tags for mass cytometry.

Mass cytometry is an emerging powerful bioanalytical technique for high-dimensional single-cell analysis. In this technique, cells are stained with metal-isotope-tagged antibodies and are analyzed by an inductively coupled plasma time-of-flight mass spectrometer. While there are more than 100 stable isotopes available in the / 75 to 209 detection range of the instrument, only about 50 parameters can be measured per cell because current reagents are metal-chelating polymers with pendant aminocarboxylate chelators that only bind hard metal ions such as the rare earths and Bi.

A Silica Coating Approach to Enhance Bioconjugation on Metal-Encoded Polystyrene Microbeads for Bead-Based Assays in Mass Cytometry.

Bead-based assays in flow cytometry are multiplexed analytical techniques that allow rapid and simultaneous detection and quantification of a large number of analytes from small volumes of samples. The development of corresponding bead-based assays in mass cytometry (MC) is highly desirable since it could increase the number of analytes detected in a single assay. The microbeads for these assays have to be labeled with metal isotopes for MC detection.

Establishing CD19 B-cell reference control materials for comparable and quantitative cytometric expression analysis.

In the field of cell-based therapeutics, there is a great need for high-quality, robust, and validated measurements for cell characterization. Flow cytometry has emerged as a critically important platform due to its high-throughput capability and its ability to simultaneously measure multiple parameters in the same sample. However, to assure the confidence in measurement, well characterized biological reference materials are needed for standardizing clinical assays and harmonizing flow cytometric results between laboratories.

Tantalum Oxide Nanoparticle-Based Mass Tag for Mass Cytometry.

Mass cytometry (MC) is a bioanalytical technique that uses metal-tagged antibodies (Abs) for high-dimensional single-cell immunoassays. Currently, this technology can measure over 40 parameters simultaneously on individual cells using metal-chelating polymer (MCP) based reagents. However, MC can in principle detect up to 135 parameters with the development of new elemental mass tags.

Metal-Encoded Polystyrene Microbeads as a Mass Cytometry Calibration/Normalization Standard Covering Channels from Yttrium (89 amu) to Bismuth (209 amu).

Mass cytometry (MC) measures metal isotope signals from single cells and bead samples. Since large numbers of isotopes can be employed as labels, mass cytometry is a powerful analytical technique for multiparameter cytometric assays. The calibration protocol in MC is a critical algorithm, which employs metal-encoded microbeads as an internal standard to correct the data for instrumental signal drift.

Tellurium-based mass cytometry barcode for live and fixed cells.

Mass cytometry is a revolutionary technology that allows for the simultaneous quantification of >40 different biomarkers with cellular resolution. The biomarkers are detected using metal-labeled antibodies as well as small-molecule probes of cell size, viability, and biochemical status. Barcoding is an important component of sample preparation because it reduces processing time, eliminates sample-to-sample variation, discriminates cell doublets, reduces the amount of antibody needed, and conserves sample.

Simultaneous Detection of Protein and mRNA in Jurkat and KG-1a Cells by Mass Cytometry.

Mass cytometry uniquely enables high-dimensional single-cell analysis of complex populations. This recently developed technology is based on inductively coupled time-of-flight mass spectrometry for multiplex proteomic analysis of more than 40 markers per cell. The ability to characterize the transcriptome is critical for the understanding of disease pathophysiology, medical diagnostics, and drug discovery.

Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells.

The diversity of nucleic acid sequences enables genomics studies in a highly multiplexed format. Since multiplex protein detection is still a challenge, it would be useful to use genomics tools for this purpose. This can be accomplished by conjugating specific oligonucleotides to antibodies.

Dual-purpose polymer labels for fluorescent and mass cytometric affinity bioassays.

We describe the synthesis and characterization of a family of poly(N-alkylacrylamide) polymers carrying 2-6 fluorescent dye molecules, ∼70 pendant DTPA (diethylenetriaminepentaacetic acid) groups, and an orthogonal maleimide end-group for covalent attachment to an antibody (Ab). These dual-purpose labels were designed for use in multiplexed immunoassays based on both mass cytometry and fluorescent flow cytometry. A challenge in the polymer synthesis was finding conditions for attaching a sufficient number of dye molecules to each polymer chain.

Metal-chelating polymers by anionic ring-opening polymerization and their use in quantitative mass cytometry.

Metal-chelating polymers (MCPs) are important reagents for multiplexed immunoassays based on mass cytometry. The role of the polymer is to carry multiple copies of individual metal isotopes, typically as lanthanide ions, and to provide a reactive functionality for convenient attachment to a monoclonal antibody (mAb). For this application, the optimum combination of chain length, backbone structure, end group, pendant groups, and synthesis strategy has yet to be determined.

Curious results with palladium- and platinum-carrying polymers in mass cytometry bioassays and an unexpected application as a dead cell stain.

We describe the synthesis of metal-chelating polymers (MCPs) with four different pendant polyaminocarboxylate ligands (EDTA, DTPA, TTHA, DOTA) and an orthogonal end-group, either a fluorescein molecule or a bismaleimide linker for antibody attachment. Polymer characterization by a combination of (1)H NMR, UV/vis absorption measurements, and thermal gravimetric analysis (TGA) indicated that each chain of the fluorescein-terminated polymers contained one dye molecule. These polymer samples were loaded with three different types of lanthanide ions as well as palladium and platinum ions.

Smart polymer nanoparticles designed for environmentally compliant coatings.

We describe the synthesis, characterization, and film-forming properties of two-component nanoparticles that undergo a reversible morphology transformation in water as a function of pH. The particles consist of a high molecular weight acrylate copolymer and an acid-rich oligomer designed to be miscible with the polymer when its -COOH groups are protonated. Attaching a fluorescence resonance energy transfer (FRET) pair to components inside the nanoparticles enabled us to assess morphology at the molecular level.

Synthesis of a functional metal-chelating polymer and steps toward quantitative mass cytometry bioassays.

We describe the synthesis and characterization of metal-chelating polymers with a degree of polymerization of 67 and 79, high diethylenetriaminepentaacetic acid (DTPA) functionality, M(w)/M(n) ≤ 1.17, and a maleimide as an orthogonal functional group for conjugation to antibodies. The polymeric disulfide form of the DP(n) = 79 DTPA polymer was analyzed by thermogravimetric analysis to determine moisture and sodium-ion content and by isothermal titration calorimetry (ITC) to determine the Gd(3+) binding capacity.

Size of bicelle defects probed via diffusion nuclear magnetic resonance of PEG.

Diffusion of various poly(ethylene glycol) (PEG) tracers of well-defined molecular weight and narrow polydispersity confined within the aqueous interstices between positively magnetically aligned bicelles was measured using pulsed-field-gradient (1)H nuclear magnetic resonance. The bicelles consisted of mixtures of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), and dihexanoylphosphatidylcholine (DHPC) in the molar ratios q = [100 DMPC +5 DMPG]/[DHPC] = 3.5, 4.