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Cytof® Mass Cytometer Discover More CyTOF® Mass Cytometer Discover More. It’s Elemental. Cellular analysis on an Familiar workflow, unprecedented scale more data 1 Cell Surface 2 Intracellular The CyTOF® Advantage Sample Preparation Immunostaining Immunostaining Incubate cells with Fix cells 375 pc metal tagged antibodies 375 pc • Simple, highly multiplexed single cell analysis Block non-specific Wash binding groups 375 pc • Robust and reproducible performance 375 pc Fix Cells Stain with metal • Absolute quantitation of target molecules 375 pc tagged antibodies 375 pc • Easy, convenient workflow Incubate with a metallointercalator2 Wash 375 pc 375 pc ® Wash Fix Cells The CyTOF Mass Cytometer introduces the next generation of 375 pc highly multiplexed single cell analyzers which will afford new insights into the biology of complex cellular networks1. Sample Processing Incubate with a metallointercalator Unlike traditional flow cytometry where detection of multiple probes is hindered by the physical limitations of fluorescence emission spectral overlap, the CyTOF instrument uses atomic mass spectrometry to allow simple, independent detection of up to 33 metal labeled antibodies (and two DNA intercalators) based ® on currently available reagents. The instrument has the capacity Introduce individual samples into the CyTOF Mass Cytometer (similar to a flow cytometer) to measure 100 independent signals and new reagents will be 375 pc introduced to take advantage of its full capability in the future. Collect data. 5 The CyTOF system is the first inorganic mass spectrometer (Analyzes 10 cells in four minutes, regardless of the number of tag elements.) specifically built for biologists and single cell applications. The inductively coupled plasma mass spectrometer at its heart Data Analysis has been used extensively by analytical chemists and is proven in the quantification of elements and stable isotopes in a variety of complex matrices. The application of this technology to multi-parametric single cell analysis brings robust and reliable Raw data is processed to give a signal strength for each analyte analytical performance to a powerful, enabling new application. represented by both an integrated analog intensity value and an integrated number of counted ions. Data output is similar to a typical flow cytometry experiment and can be exported as text and FCS3.0 formats to be fully compatible with flow cytometry data processing software. Simple, highly multiplexed Robust and reproducible single cell analysis performance 100 14 elements: 30 isotopes Er Tm Up to 100 independent Ba Ho Yb Lu Reproducibility between Absolute quantification of Nd Tb 90 Sm Dy La Gd detection channels 80 Pr Eu samples, between systems target molecules 70 The unique multiplex analysis capability 60 The use of atomic mass spectroscopy is The CyTOF instrument offers the specificity, 50 of the CyTOF® system is already enabling well established in the field of elemental dynamic range and quantitative capability of 40 ® new insights into the functional complexity 30 analytical chemistry and the CyTOF atomic mass spectrometry in a format that of biological systems at the single cell signal intensity 20 instrument will deliver robust and is familiar to flow cytometry practitioners. level. A recent example published in 10 reproducible performance from the first The quantitative aspect of flow cytometry is Science3 describes how the technology 0 day of installation in your laboratory. Data currently underutilized primarily due to was used with an immunophenotyping 138 143 148 153 158 163 168 173 178 generated is highly reproducible with a low absence of independent quantitative panel 31 labeled antibodies to provide a (stable isotope) atomic mass coefficient of variation between technical analytical methods and lack of standard uniquely detailed view of cell differentiation replicates, between systems and between materials. Mass cytometry offers a unique 138-178 segment of mass spectrum for a homogeneous sample of several in the human hematopoietic system. enriched isotopes of lanthanides. The complete separation of detection users. This characteristic makes its use opportunity to simultaneously analyze more The CyTOF technology currently enables signals allows selection of any combination of labels to be used in multiplexed particularly well suited to multi-site or subtle quantitative changes in intracellular single cell analysis. analysis up to 100 stable isotope labels in longitudinal studies. and membrane bound proteins within a single sample. Currently, DVS Sciences complex cell populations. Inc. provides labeling kits for 33 different CD14 Tm169 metal tags and will increase the range labeled antibody or as many as 33. In CD45 Tb159 CD66 Dy164 CD45 ABC Summary Sample #1 Sample #2 Sample #3 Average Std Dev in the future to fully utilize the multiplex addition, with effectively no overlap CD4 Nd142 CD16 Nd144 CD45-AF700 ABC 215,447 177,569 269,865 220,960 37,881 detection capabilities of the instrument. between atomic mass signals, inherent CD8a Nd146 by Flow Cytometry CD20 Gd156 Because the mass spectrometer provides compensation problems are completely CD45-AF700-Tb ABC CD11b Gd158 189,050 209,615 187,245 195,303 10,147 by Mass Cytometry at least 3 orders of magnitude resolution eliminated. For the first time, complex HLA-DR Er166 CD13 Er170 between adjacent detection channels, multi-parametric cytometry is within easy %, without RBC & debris CD15 Sm147 compensation is not required. Additionally, reach of any life science laboratory. CD45RO Sm152 300,000 CD3 Pr141 CD33 Ho165 the sensitivity is practically the same 200,000 CD38 Yb171 across all channels measured and as a In contrast, the limitations of fluorescence CD19 Yb176 consequence, the combination of labels signal overlap significantly restricts the CD56 Eu151 100,000 CD44 Eu153 used in an experiment can be essentially number of simultaneous probes that can be CD45RA arbitrary. Designing experiments is simple used, especially when proteins are present lr 191 lr 193 Sample #1 Sample #2 Sample #3 whether you are using as little as a single at very different concentrations. CD45-AF700 ABC by Flow Cytometry CD45-AF700-Tb ABC by Mass Cytometry Seven aliquots of CB were analyzed on seven consecutive days. Ten gated In the example above, flow cytometry and mass cytometry were compared using populations were established based on the markers listed. the same affinity probes on the same cell samples. The data generated on each platform generally agrees with published literature for CD45 expression values on normal lymphocytes. A key component in mass cytometry quantification system is the accurate determination of the number of metal atoms attached to each antibody molecule. Utilization of multiple lanthanide labeled antibodies is robust because no compensation matrix is required. Proven technology, Everything you need novel application at your fingertips CyTOF® Mass Cytometry MAXPAR® Antibody The CyTOF instrument is based on the composition of each cell is separately Labeling Kits application of robust and proven atomic analyzed. In a typical cell analysis mass spectrometry uniquely applied to experiment, four minutes of raw data single cell analysis. Samples containing collection is sufficient for analysis of 105 Rapid, easy preparation of Antibody conjugation metal-labeled cells are introduced cells independent of the number of tag individually into an Inductively Coupled elements. labeled antibodies to MAXPAR metal tag Plasma, were the cells are atomized and MAXPAR labeling reagent kits for 32 ionized. The atomic ions are extracted It is important to note that cells without any different metals are available from DVS into the ion optics and time-of-flight tagging elements cannot be detected by Sciences Inc. for preparation of labeled regions where they are separated by mass mass cytometry: Only element tags can antibodies or intracellular proteins. Kits and counted. The elemental signature be registered with high sensitivity and are optimized and quality control tested of the cell includes the element tags specificity, and therefore there is no “auto- for use with the CyTOF® instrument and introduced with the antibodies and fluorescence”-like effects. Elemental tags are available in pack sizes sufficient to metallointercalators. The presence of the are chosen from rare elements whose label up to 400ug or up to 4mg of tag element indicates that the antibody natural concentration in a biological sample antibody. Labeling methods are simple found and bound the target biomarker, is below the detection limit. Unstained cells and convenient and require only 2.5 hours and the intensity of the signal is directly are “transparent” to the mass cytometer. A to complete. The labeling kits utilize a proportional to the number of antibodies complete description of the instrument can traditional approach of adding a metal bound per cell (ABC). The elemental be found in Bandura et al, 20094. tag using disulfide reduction (typically in A – Lanthanide loading of metal-chelating polymer in 3K MWCO spin filter. B – Antibody reduction with TCEP in 50K MWCO spin filter. the Fc region of the antibody) through a C – Incubation of metal-containing polymer tag with reduced antibody in the 50K MWCO spin filter. RF load coil ion cloud maleimide linker. Labeled antibodies are stable for approximately 6 months after preparation. time pc pc pc 375 375 375 intensity interface injector Ar plasma mass mass Data acquisition process in the mass cytometer. The cell is atomized and
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