2014

Sean Bendall: Mass Cytometry: Guilt-free, 35-plus Parameter, Single Cell Analysis for Proteomic Dissection of Immune Function

Classical four-color fluorescence flow cytometry helped define the major cell subsets of the immune system that we understand today (i.e. T-cells, B-cells, macrophages). Machines with eight or more colors brought characterization of rare immune subsets and stem cells. With intracellular staining, higher parameter measurements lead to examination of regulatory signaling networks and patient stratification with clinical outcomes. However, this progression has now been stymied by the limit of fluorescence parameters measurable, realistically capped at 12-15 due to boundaries in instrumentation and spectral overlap considerations in fluorophore-based tagging methods. Now, a novel combination of elemental mass spectrometry with single cell analysis (mass cytometry) offers examination of 30-50 parameters (theoretically up to 100) without fluorescent agents or interference from spectral overlap. Instead, it utilizes non-biological, elemental isotopes as reporters. By exploiting the resolution, sensitivity, and dynamic range of elemental mass spectrometry, on a time-scale that allows the measurement of 1000 individual cells per second, this device offers a much-simplified alternative for ultra-high content cytometric analysis. At Stanford, using the world’s first commercial version of this instrument (CyTOF), we have applied simple modifications to protocols already established in our lab for quantization of cellular signaling events in immunological subtypes. Already measuring 20+ intracellular antigens (phosphorylations) in conjunction with 10+ cell surface markers we detail the approaches we are taking towards an unprecedented profile of cytokine/immune responses of all major cell types in human blood and bone marrow. We will present these studies and demonstrate the detailed systems-level view of immune function they reveal.

http://www.youtube.com/watch?v=Uk8d20EKORM

Wendy Fantl: What it Takes to Perform a Mass Cytometry Experiment: The Devil is in the Details!

The excitement of being able to measure upwards of 40 parameters simultaneously on a cell-by-cell basis should not be a pretext for relaxing the criteria for experimental rigor. While the mass cytometer is, of course the “Prima Donna” (in the experiment!), the pivotal role of you, the researcher, demands very significant and often overlooked preparation-time before “pressing the button”! This talk will examine many of the variables that must be considered for an experiment in order to obtain meaningful data. I will discuss how experimental design and reagent optimization and validation take up far more time than running samples on the machine. The consequent dense datasets are comprised of measurements of 40 parameters per single-cell for hundreds of thousands to millions of cells again necessitating allocation of far more time for number crunching than is ever required for running samples through the machine. The overall message of this presentation is to provide an insight into the very considerable demands on time and expertise that is required of this powerful analytical technique.  

http://youtu.be/CCAOvxxTJio

Matt Spitzer: Tools of the Trade: Reagent Development and Antibody Conjugation  A brief overview of CyTOF antibody chelation chemistry will be covered. A summary of the antibody conjugation protocol will be provided as well as information regarding intercalating reagents, viability stains and barcode reagents. I will also address relevant troubleshooting and optimization topics including titrations, antibody purification and approaches for circumventing conjugation for problematic antibody clones.  

http://youtu.be/lluoAdlJIdI

TJ Chen (Director of Informatics, Cytobank, Inc): Cytobank - Manage, Analyze and Share Flow Cytometry Data from Anywhere  Mass and fluorescent phosphoflow cytometry generate a unique challenge in data analysis, since they require that quantitative measurements are made on multi-dimensional data. Therefore, we have written a software platform called Cytobank, that enables: 1) secure storage of annotated flow cytometry data, 2) sharing of data between users and collaborators, eliminating the need for shared drives, 3) annotation and tagging of data and individual experiments, 4) compensation 5) calculation and export of custom statistics readable by software including Excel, 6) heatmap, histogram, and 2D plot generation, 7) pivoting of data to visualize many different parameters rapidly and easily, and 8) advanced visualization and annotation tools. We will present the background and development of Cytobank, as well as show a live demo of the software.

https://www.youtube.com/watch?v=9OuQ8hgsqes

Greg Behbehani M.D, Ph.D: Cell cycle analysis using mass cytometry  This presentation will discuss the basic methods for analyzing the cell cycle by mass cytometry. The current methodology relies on Incorporation of 5- iodo-2-deoxyuridine (IdU) to label S-phase cells, and cyclins A and B1 to separate G1 from G2 cells. G0 cells can be identified using an antibody against retinoblastoma protein phosphorylated at serines 807 and 811, and M phase cells are detected through the use of an antibody directed against histone H3 phosphorylated at serine 28. These methods yield equivalent results to traditional fluorescence methods in both cultured cell lines and stimulated normal T cells. This analysis can be combined with large panels of surface or functional markers to measure the cell cycle across multiple sub-populations of cells within complex samples.  

https://www.youtube.com/watch?v=YYZCp877KyQ

Kara Davis: Understanding Acute Lymphoblastic Leukemia by Taking Cues from Normal B cell Development  This presentation will discuss how the application of mass cytometry has allowed a better understanding of human B cell lymphopoiesis. Then I will discuss how this serves as a foundation to build a novel model of a common B cell malignancy, acute lymphoblastic leukemia

https://www.youtube.com/watch?v=Jb8bhI8sLrA

Eli Zunder: Kinase Inhibitor Profiling With Mass-Tag Cell Barcoding

Mass cytometry enables quantitative, high-content analysis at the single cell level with more measured parameters than traditional fluorescence-based flow cytometry. Here I describe a method termed mass-tag cellular barcoding (MCB) that significantly increases sample throughput by multiplexing masstag encoded samples while enabling comprehensive signaling network analysis. 96-well format MCB was used to characterize the signaling dynamics of human peripheral blood mononuclear cells (PBMCs) and to define the impact of 24 commonly used small molecule kinase inhibitors on this system. For each small molecule, 14 phosphorylation states were measured per cell in 14 PBMC types under 96 conditions, resulting in 18,816 quantified phosphorylation levels from a single multiplexed sample.

https://www.youtube.com/watch?v=xIL-ei9ehHQ

Pier Federico Gherardini: Visualization and analysis of high-dimensional single-cell datasets

This presentation will serve as a primer for the visualization and analysis of high-dimensional sing-cell datasets. After having addressed some basic definitions, we will identify the fundamental computational issues involved and detail the workings of SPADE and other methods recently developed in the lab.

https://www.youtube.com/watch?v=__Yhl1I7Uy4

Robert Bruggner: Overview of SPADE and Citrus Software I’ll briefly cover two software tools for working with flow cytometry data. First we’ll cover running SPADE on the Cytobank website. Time permitting; we’ll also go over a demo of the Citrus software used for detecting stratifying cell subsets.

https://www.youtube.com/watch?v=fLCzQahrFMA

Holden Maecker (Director, Human Immune Monitoring Center): New technologies in the Human Immune Monitoring Center  The Stanford Human Immune Monitoring Center was conceived as a “one-stop shop” for immune monitoring platforms, including technology development, and data mining. This presentation will review the need for such a comprehensive approach to human immunology, and highlight some new additions to the HIMC technical portfolio.

https://www.youtube.com/watch?v=X4OEryPnxns

Yael Rosenberg-Hasson (Technical Director, Human Immune Monitoring Center): Multiplexed Assays for Human Cytokine Detection Multiple platforms for multiplexed immunoassays exist, of which Luminex is the most widely used. These assays use microspheres with separate fluorescent reporting of both bead address and analyte binding. They can provide picogram per milliliter sensitivity and multiplexing of 50 or more analytes in a single well, requiring <50 microliters of serum, plasma, or other fluid. Issues of matrix effects, concentration calculation, etc. will be discussed. Comparison data with an electrochemiluminescence platform (MesoScale Discovery) will also be presented.

https://www.youtube.com/watch?v=GKmRhjTfRB8

Joanna Lilienthal (Director, Translational Applications Service Center) and Alice Fan (Assistant Professor, Medicine/Oncology): Nanoimmunoassays For Phosphoprotein Analysis

The NanoPro platform enables detailed characterization of proteins from limited biological samples. Current methods of protein detection are insensitive to subtleties in post-translational modification and often require large samples size. NanoPro technology allows quantification of various protein and other small molecule isoforms using capillary electrophoresis. We have used NanoPro to analyze proteins in as little as 500 primary cells and have embraced the system to assist in evaluating clinical therapeutics. Thus, NanoPro may be a promising novel technology for new diagnostic and biomarker studies.

https://www.youtube.com/watch?v=PQ4xSC28Vm0

Arnold Han (Postdoctoral Fellow, Davis Lab): Linking T cell receptor repertoire to multi-parametric phenotyping at the single-cell level

T lymphocytes recognize a vast array of different antigens through their T cell receptor (TCR) heterodimers. They have very diverse functional activities, from stimulating B cells to make high affinity antibodies to inhibiting responsiveness. In many cases, the major specificities and functional characteristics of a T cell response are not known. Thus, we have devised a methodology by which TCR heterodimers of individual T cells can be amplified and sequenced together with genes characteristic of the different T cell types, linking function and specificity. I will discuss this approach, and show data from tumor-infiltrating lymphocytes to demonstrate its potential utility.

https://www.youtube.com/watch?v=4jnUFmC6KQk

Sanchita Bhattacharya (Scientist, Butte Laboratory): Role of bioinformatics in Immune “Omics” technologies  

Advanced immunoassays have given researchers the capability to measure the immune components in high throughput fashion. We will discuss the practical aspects of statistical experimental design to optimize the downstream analyses of the data generated from these assays. We also emphasize on how to think about issues that come up in quantifying the cell secreted cytokines though multiplex cytokine assays, such as normalization, significance estimation, and pattern mining methods.  

https://www.youtube.com/watch?v=uGwkDW3RRw0