Enabling Indium Channels for Mass Cytometry by Using Reinforced Cyclam-Based Chelating Polylysine.

The synthesis of a polylysine polymer functionalized with the previously reported astonishingly inert [In()] chelate was performed. A biotin end group allowed the conjugation to biotinylated beads by the intermediary of a fluorescein isothiocyanate/neutravidin receptor. High quality imaging mass cytometry trials, based on In detection were performed to highlight the behavior of the material.

Highly Stable and Inert Complexation of Indium(III) by Reinforced Cyclam Dipicolinate and a Bifunctional Derivative for Bead Encoding in Mass Cytometry.

H cb-te2pa, a cross-bridged cyclam functionalized by two picolinate arms, was used for the formation of an incredible inert In chelate. The inertness of the complex was evaluated by UV/Vis experiments in several competitive media and was highlighted by the comparison with [In(dota)] and [In(dtpa)] (H dota = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, H dtpa = diethylenetriamine pentaacetic acid), which are currently used in biological applications. For the first time, a bifunctional analogue of H cb-te2pa was prepared by C-functionalization to keep its coordination properties intact.

Imaging Mass Cytometry.

Imaging Mass Cytometry (IMC) is an expansion of mass cytometry, but rather than analyzing single cells in suspension, it uses laser ablation to generate plumes of particles that are carried to the mass cytometer by a stream of inert gas. Images reconstructed from tissue sections scanned by IMC have a resolution comparable to light microscopy, with the high content of mass cytometry enabled through the use of isotopically labeled probes and ICP-MS detection. Importantly, IMC can be performed on paraffin-embedded tissue sections, so can be applied to the retrospective analysis of patient cohorts whose outcome is known, and eventually to personalized medicine.

Biodistribution of cisplatin revealed by imaging mass cytometry identifies extensive collagen binding in tumor and normal tissues.

Imaging mass cytometry was used for direct visualization of platinum localization in tissue sections from tumor and normal tissues of cisplatin-treated mice bearing pancreas cancer patient-derived xenografts. This recently-developed technology enabled simultaneous detection of multiple markers to define cell lineage, DNA damage response, cell proliferation and functional state, providing a highly detailed view of drug incorporation in tumor and normal tissues at the cellular level. A striking and unanticipated finding was the extensive binding of platinum to collagen fibers in both tumor and normal mouse tissues.

Single-cell measurement of the uptake, intratumoral distribution and cell cycle effects of cisplatin using mass cytometry.

Although of fundamental importance to the treatment of cancer patients, the quantitative study of drug distribution and action in vivo at the single cell level is challenging. We used the recently-developed technique of mass cytometry to measure cisplatin uptake into individual tumor cells (Pt atoms/cell), combined with measurement of the rate of IdU incorporation into DNA (I(127) atoms/cell/min) and tumor hypoxia identified by the 2-nitroimidazole EF5 in cisplatin-treated BxPC-3 and ME-180 xenografts. Pt levels of 10(5) to 10(6) atoms/cell were obtained following a single cisplatin treatment using clinically relevant doses.

An introduction to mass cytometry: fundamentals and applications.

Mass cytometry addresses the analytical challenges of polychromatic flow cytometry by using metal atoms as tags rather than fluorophores and atomic mass spectrometry as the detector rather than photon optics. The many available enriched stable isotopes of the transition elements can provide up to 100 distinguishable reporting tags, which can be measured simultaneously because of the essential independence of detection provided by the mass spectrometer. We discuss the adaptation of traditional inductively coupled plasma mass spectrometry to cytometry applications.

Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum.

Flow cytometry is an essential tool for dissecting the functional complexity of hematopoiesis. We used single-cell "mass cytometry" to examine healthy human bone marrow, measuring 34 parameters simultaneously in single cells (binding of 31 antibodies, viability, DNA content, and relative cell size). The signaling behavior of cell subsets spanning a defined hematopoietic hierarchy was monitored with 18 simultaneous markers of functional signaling states perturbed by a set of ex vivo stimuli and inhibitors.

Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry.

A novel instrument for real time analysis of individual biological cells or other microparticles is described. The instrument is based on inductively coupled plasma time-of-flight mass spectrometry and comprises a three-aperture plasma-vacuum interface, a dc quadrupole turning optics for decoupling ions from neutral components, an rf quadrupole ion guide discriminating against low-mass dominant plasma ions, a point-to-parallel focusing dc quadrupole doublet, an orthogonal acceleration reflectron analyzer, a discrete dynode fast ion detector, and an 8-bit 1 GHz digitizer. A high spectrum generation frequency of 76.

Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry.

Advances in the development of highly multiplexed bio-analytical assays with inductively coupled plasma mass spectrometry (ICP-MS) detection are discussed. Use of novel reagents specifically designed for immunological methods utilizing elemental analysis is presented. The major steps of method development, including selection of elements for tags, validation of tagged reagents, and examples of multiplexed assays, are considered in detail.

Messenger RNA Detection in Leukemia Cell lines by Novel Metal-Tagged in situ Hybridization using Inductively Coupled Plasma Mass Spectrometry.

Conventional gene expression profiling relies on using fluorescent detection of hybridized probes. Physical characteristics of fluorophores impose limitations on achieving a highly multiplex gene analysis of single cells. Our work demonstrates the feasibility of using metal-tagged in situ hybridization for mRNA detection by inductively coupled plasma mass spectrometry (ICP-MS).