Enhanced multiplexing in mass cytometry using osmium and ruthenium tetroxide species

Abstract

Mass cytometry facilitates high-dimensional, quantitative, single-cell analysis. The method for sample multiplexing in mass cytometry, called mass-tag cellular barcoding (MCB), relies on the covalent reaction of bifunctional metal chelators with intracellular proteins. Here, we describe the use of osmium and ruthenium tetroxides (OsO 4 and RuO 4 ) that bind covalently with fatty acids in the cellular membranes and aromatic amino acids in proteins. Both OsO 4 and RuO 4 rapidly reacted and allowed for MCB with live cells, crosslinked cells, and permeabilized cells. Given the covalent nature of the labeling reaction, isotope leaching was not observed. OsO 4 and RuO 4 were used in a 20-sample barcoding protocol together with palladium isotopes. As mass channels occupied by osmium and ruthenium are not used for antibody detection the number of masses effectively utilized in a single experiment is expanded. OsO 4 and RuO 4 can therefore be used as MCB reagents for a wide range of mass cytometry workflows. V C 2016 International Society for Advancement of Cytometry Key terms mass tag cellular barcoding; CyTOF; mass cytometry; osmium tetroxide; ruthenium tetroxide ELEMENTAL mass spectrometry-based cytometry, or mass cytometry (1-4), is a recently developed technology platform that enables characterization of single cells using affinity reagents, such as antibodies covalently linked to polymeric, metalloaded chelators (4). Use of elemental metal isotopes, as opposed to fluorochromes, enables quantification of up to 46 epitope abundances simultaneously on individual cells (1-3). The detection modality in mass cytometry is an inductively coupled plasma time of flight mass spectrometer (ICP-TOF MS). In this instrument, metal isotope labeled cells are atomized and ionized in an argon plasma and the abundance and distribution of the isotopes is measured in a TOF mass analyzer. With the current generation of mass cytometry instruments, 135 isotope channels can be recorded simultaneously at an acquisition rate of $2,000 cells/ second (5,6).Recently we introduced a multiplexing strategy called MCB, in which metal isotopes are used to label samples with unique metal isotope barcodes (6,7). After sample mixing, cells are labeled with antibodies and analyzed on the mass cytometer. In a first data analysis step, the single cells are then assigned to their sample based on their metal isotope barcode. The main advantages of this approach are that all cells are stained homogeneously with the same antibody mix, the fluidics system of the mass cytometer does not have to be cleaned for individual samples, and all MCB samples are subjected to the same instrument drift. Using this strategy, up to 96 samples can be barcoded and 39 different parameters analyzed on each single cell (7).Both lanthanide isotopes and palladium isotopes have been used to barcode cells (6,7). In both methods ion chelating bifunctional compounds that react with cells

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

“…Usually, sample barcoding is achieved by covalently labelling cellular proteins with dyes or mass tags via reactive thiols or primary amines [70,72,176,177,181], or by antibodies [61,71,178,182]. In mass cytometry, lipid-reactive RuO 4 and OsO 4 have also been demonstrated as applicable for barcoding [180].…”

Titrating Complex Mass Cytometry Panels

“…Mass cytometry enables the simultaneous measurement of over 40 antigens on single cells using metal isotope conjugated antibodies, generating highly complex datasets with minimal experimental artifacts . As the number of antibodies used to investigate biologically heterogeneous cells increases, so do the demands for an efficient and thorough approach to determine optimal antibody titers.…”

Single‐Cell Mass Spectrometry Approaches to Explore Cellular Heterogeneity

“…This approach enables the simultaneous analysis of over 30 cellular features, for example,the presence of particular receptors,for millions of cells at ar ate of around 2000 cells s À1 . [109][110][111] Here,w e present some of the recent single-cell applications of this technique. [108] Mass cytometry represents at ransition between targeted and untargeted methods,w here fewer than 10 and more than 100 analytes, respectively,a re identified.…”

“…Recent advances in mass cytometry have been discussed in detail elsewhere. [109][110][111] Here,w e present some of the recent single-cell applications of this technique.…”