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. 1 µm2 spots of a tissue section. The plume is then transferred to the CyTOF, generating an image of biomarker expression. Similar measurements are possible with multiplexed ion bean imaging (MIBI). The unit mass resolution of the ICP-TOF-MS detector allows for multiparametric analysis of (in principle) up to 130 different parameters. Currently available reagents, however, allow simultaneous measurement of up to 50 biomarkers. As new reagents are developed, the scope of information that can be obtained by mass cytometry continues to increase, particularly due to the development of new small molecule reagents which enable monitoring of active biochemistry at the cellular level. This review summarizes the history and current state of mass cytometry reagent development and elaborates on areas where there is a need for new reagents. Additionally, this review provides guidelines on how new reagents should be tested and how the data should be presented to make them most meaningful to the mass cytometry user community.

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 NaYF4:Yb3+/Er3+ NPs. A two-step ligand exchange was used to coat NP surfaces with either methoxy-PEG2K-neridronate (PEG-Ner) and/or poly(sulfobetaine methacrylate)-neridronate (PSBMA-Ner). Both modifications provided long-term colloidal stability in PBS buffer. IMC measurements on tonsil tissue showed that PSBMA-Ner or a 1:1 mixture of PSBMA-Ner + PEG-Ner effectively suppressed nonspecific binding (NSB) at 2 × 1010 NPs/mL, unlike PEG-Ner alone. However, breast cancer tissue samples showed increased NSB at titers above 2 × 1010 NPs/mL. Reduced NSB with mixed PEG-Ner and PSBMA-Ner coatings opens the door for using heterobifunctional PEGs for the development of NP conjugates with bioaffinity agents, enabling more sensitive and specific MC analyses.

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). We recently reported a new class of metal-chelating polymers with dipicolylamine (DPA) chelators that could bind Re and Pt. They also showed significant levels of NSB. Here, to reduce the NSB of the Pt-DPA polymer, we grafted water-soluble oligomers to the distal end of the dipicolylamine pendant group. Methoxy(polyethylene glycol) (DP = 24) was effective as was poly(sulfobetaine methacrylate) (DP = 29). Reacting the Pt-Cl bond of the metalated polymer with glutathione was remarkably effective at suppressing NSB. These results open the door to Pt-isotope-based metal-chelating polymers as new mass tags for mass cytometry.

Gas chromatographic fingerprint analysis of secondary metabolites of Stachys lanata (Stachys byzantine C. Koch) combined with antioxidant activity modelling using multivariate chemometric methods.

S. lanata has been traditionally used as a medicinal plant due to its various biological activities such as antioxidant activity. Therefore, identification and quality control studies of this plant are of great importance. To this end, gas chromatography (GC) combined with chemometrics was proposed for fingerprint analysis of S. lanata samples. This study sought to classify GC fingerprints of twenty-eight S. lanata samples from eight different regions of Iran and more importantly, to correlate fingerprints to the antioxidant activity to select S. lanata volatile antioxidant markers. S. lanata samples were classified into five and three classes using partial least squares-discriminant analysis (PLS-DA) according to their GC fingerprints and antioxidant peaks, respectively. The results of PLS regression (PLS-R) and variable importance in projection (VIP) showed that phenol, 2,4-bis (1,1-dimethylethyl)-, hexadecanoic acid- ethyl ester, vitamin E, Beta- sitosterol, and 1- monolinoleoylglycerol trimethylsily ether are volatile antioxidant markers of S. lanata samples.