To quantify peptide conjugation efficiency, 20?l beta-mercaptoethanol (14.3?M stock solution) and 20?L DMF were added to an aliquot (0.1?mg in 60?l H2O) of purified MUSOT-peptide conjugates and allowed to react for 48?h on a shaker at 25?C. plasma mass spectrometry (LA-ICP-MS) enables quantitation of metallic contents in the single-cell level with additional insights on sub-cellular localization of NPs, however this image-based method also suffers from low throughput (tens to hundreds of cells typically analysed) and relatively low level of sensitivity (requiring millions of atoms per cell)15,16,17. Solitary particle ICP-MS (SP-ICP-MS) is definitely another ICP-based method that utilizes time-resolved mode to enable direct quantification of the number concentration, size distribution of NPs, and their state of agglomeration18,19. It Versipelostatin has allowed for single-cell analysis of metal-containing cells when the cell concentration was cautiously optimized to avoid overlapping cells in the detector20,21. However, SP-ICP-MS is only suitable for NPs larger than 20?nm in diameter and is usually coupled with additional methods such as LA-ICP-MS to determine NP cellular distribution and quantitation22. Currently you will find no label-free measurement techniques that can quantify inorganic nanomaterials Versipelostatin of arbitrary size/chemistry in solitary cells at high throughput11. Mass cytometry is definitely a recently developed method merging time-of-flight ICP-MS with circulation cytometry23. Single-cell suspensions are labelled with metallic isotope-tagged antibodies or additional binding probes. Individual cells are then ionized in an argon plasma followed by time-of-flight mass spectrometry, which enumerates each metallic species present in the producing ion cloud24,25. Current Helios mass cytometry devices permit up to 50 metallic isotope labels (atomic weights ranging from 75 to 209) to be detected simultaneously on a single cell. Such highly multiparametric detection offers offered fresh insights into the difficulty of biology, in applications ranging from deep phenotyping of tumours to immune system signalling pathways26,27. Here we display for the first time that when combined with nanoparticle calibration, mass cytometry can also be used as a powerful fluorophore label-free method to track inorganic nanoparticles in tandem with highly multivariate cellular phenotyping, enabling quantitative analysis of the fate of inorganic Versipelostatin nanomedicines. Using platinum NPs (AuNPs) as a representative inorganic nanomaterial with relevance for varied biomedical applications6,7,28,29,30,31,32, we demonstrate the capacity of mass cytometry to enumerate nanoparticles in individual cells having a level of sensitivity Kcnh6 orders of magnitude greater than circulation cytometry. We display that mass cytometry overcomes difficulties in fluorescence-based analysis of autofluorescent cells cells, and illustrate the value of combined solitary cell NP detection with antibody-based phenotyping, using insights derived from mass cytometer analysis to select a nanoparticle composition that accumulates in dendritic cells for vaccination. Results AuNP per cell quantitation via mass cytometry We 1st synthesized AuNPs with similar inorganic core diameters but three different surface chemistries expected to have unique biodistributions and cellular uptake (Fig. 1a): 3-mercapto-1-propanesulfonate (MPSA) NPs, coated by a dense coating of short sulfonate-terminated ligands that strongly interact with water; 11-mercapto-1-undecanesulfonate/1-octanethiol (MUS/OT) NPs bearing an amphiphilic combined ligand shell, which are water soluble but strongly interact with cell membranes;33,34 and poly(ethylene glycol) NPs sterically stabilized by PEG to reduce opsonization by serum parts35. The particles were relatively monodispersed with related mean gold core diameters 2.5C4?nm and negative zeta potentials (Fig. 1b,c and Supplementary Table 1). Open in a separate windows Number 1 Platinum nanoparticle ligand chemistry and size distribution.(a) Schematics of MPSA (3-mercapto-1-propanesulfonate) coated AuNPs, MUS (11-mercapto-1-undecanesulphonate) and OT (1-octanethiol) combined ligand-coated AuNPs, and PEG (tetraethylene glycol)-coated AuNPs. (b) Representative TEM image of MUS/OT NPs (level pub 10?nm). (c) Size distributions of MPSA, MUS/OT and PEG NPs identified from TEM. Pilot experiments founded that platinum was readily recognized by mass cytometry analysis of cultured cells incubated with AuNPs using either CyTOF2 Versipelostatin or Helios devices. We 1st compared the level of sensitivity of mass cytometry and circulation cytometry for detecting NP uptake, incubating BODIPY-labelled MUS/OT NPs36,37 with Natural macrophages for 6?h, followed by circulation cytometry or mass cytometry. Calibration of the TOF detector (observe Methods) enabled a direct enumeration of platinum ions, and therefore mean numbers of nanoparticles accumulated per cell. Platinum uptake by macrophages was clearly detectable by mass cytometry across this entire concentration range (with detector saturation happening at an top detection limit of 1 1.5 106 particles per cell, Fig. 2c), whereas NPs at concentrations of 0.1?g?ml?1 or lesser were not detected in cells using circulation.