CyTOF/CODEX mini-course


Day 1: Monday, May 14, 2018

Garry Nolan: Welcome and Introduction

Sean Bendall: The Nuts and Bolts of Mass Cytometry

This lecture will cover the theory behind how mass cytometry and data acquisition work. We will review the standardization of assays, ion counting and absolute quantification, as well as highlight analytical challenges that will be addressed in subsequent lectures.

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

Pier Federico Gherardini (Parker Institute) Visualization and analysis of high-dimensional single-cell datasets

The aim of this presentation is to introduce you to some of the fundamental issues you need to keep in mind when analyzing high-dimensional single-cell data. We will start by looking at some basic definitions and concerns that are common across all the computational methods that have been developed in recent year for this task. We will then look in more detail at the inner workings of some these methods in order to give you a better understanding of what the differences and the commonalities are.

Matthew Spitzer (UCSF): Applying CyTOF to understand the mechanisms of effective anti-tumor immune responses

Mass cytometry uniquely enables us to identify every type of cell present in a complex tissue while also revealing aspects of each cell's behavior in a particular context. I will provide an overview of our applications of CyTOF-based approaches to understand immune responses to cancer and the mechanisms that mediate effective cancer immunotherapy.

Matthew Spitzer (UCSF): Applying CyTOF to understand the mechanisms of effective anti-tumor immune responses

Mass cytometry uniquely enables us to identify every type of cell present in a complex tissue while also revealing aspects of each cell's behavior in a particular context. I will provide an overview of our applications of CyTOF-based approaches to understand immune responses to cancer and the mechanisms that mediate effective cancer immunotherapy.

Day 2: Tuesday, May 15, 2018

Yury Goltsev:Multiplex Imaging of Immune and Tumor Environment

CODEX (CO-Detection by indEXing) is a compilation of multiplexed staining techniques based on DNA labeled antibodies. Antibody binding events are rendered iteratively using either just DNA barcodes, or a combination of DNA barcoding and a in-situ polymerization-based indexing procedure. I will elaborate on the variation of the technique (CODEX1) based on polymerase driven single base extension. CODEX1 has no overall limit for the total number of co-stained epitopes and allows two distinct antibodies imaged during each 10- minute indexing cycle . Fluorescent signals from multiple rounds of indexing are computationally combined into a multi-channel image stack and subjected to image segmentation and quantification. We demonstrate the performance of CODEX1 on classic suspension cells and tissue sections. When applied to tissue sections, CODEX produces multidimensional data enabling identification of both classically determined and novel cellular neighborhoods. In a use case with normal murine splenic versus a lupus model (MRL/lpr) we demonstrate CODEX’s capacity to delineate profoundly different tissue micro-architectures. In addition to systematic characterization of secondary immune organ structure we show how CODEX data reveals fundamental principles of tissue organization such as homotypic cell adhesion and impact of cellular neighbors on effector molecule expression in cells. Using mouse models of lymphomas and hepatocellular carcinomas induced by overexpression of myc and a combination of myc and twist we demonstrate how CODEX can be applied to study the architecture and microenvironment of immune infiltrates. The CODEX platform has a potential to expand the range of diagnostic antigen co-detection in clinical and research settings and opens a path towards automated deep proteomic analysis of complex cellular compartments in normal and clinically aberrant samples.

Christian Schuerch:CODEX: Technology overview and application

CODEX (CO-Detection by antibody indEXing) is a fluorescence based multiplexed imaging platform that can measure more than 50 parameters at single-cell resolution within intact tissue. Standard immunofluorescence assays are limited to just 2-3 parameters due to spectral overlap. In CODEX, specialized DNA-tagged antibodies and iterative cycles of adding and removing fluorescence-labeled complementary oligonucleotides using a microfluidics system allow acquiring the high-parameter data. In this talk, I will give an overview of the CODEX technology, an introduction into the chemistry of DNA-conjugated antibodies, inputs for antibody panel development and validation, and will briefly discuss how CODEX data are analyzed. In addition, I will highlight recent developments for CODEX in formalin-fixed, paraffin-embedded tissue.

Nikolay Samusik:Computational analysis of CODEX data

Computational analysis of CODEX data is an involved and multi-stage process. The preparatory phase includes raw data processing (drift-compensated, deconvolved, cropped), the processed images are subject to segmentation and positional spillover compensation, resulting in spatially enabled FCS files. Downstream analysis involves data visualization, phenotypic classification of cells, mapping of cell phenotypes back to the image and analysis of i-niche neighborhoods. Nikolay will cover the entire process, explaining the use of various computational tools at each step and fine-tuning of parameters

Salil Bhate:Advanced concepts in high-parameter tissue analysis

The aim of this session is to highlight novel ways to think about tissue imaging data, and emerging analytical strategies for interrogating it. Technical discussion of algorithms will be limited. We will cover: Neural networks, Cellular communities and motifs, Multiscale analysis, and Dynamics from static images

Sizun Jiang:Multiplexed Ion Beam Imaging (MIBI) applications to basic and translational biology

Multiplexed Ion Beam Imaging (MIBI) is a new imaging modality that harnesses the resolution and sensitivity of secondary ion mass spectrometry (SIMS) with elemental mass tagged antibodies (similar to CyTOF) to simultaneously visualize 40-50 targets in tissues and cells. Here, I will present some of the capabilities of MIBI in tissue and cellular imaging, from 50 - 300nm X Y resolutions. I will introduce some of the analytical tools and pipelines for MIBI data analysis, and show examples of the applications of MIBI to basic biology and translational research. Finally, I will give a brief overview of current directions for MIBI in the laboratory for novel tools in multi parametric imaging.

Day 3: Wednesday, May 16, 2018

Holden MaeckerNew technologies in the Human Immune Monitoring Center

This presentation will highlight the value of comprehensive immune monitoring, the way that this has been organized at Stanford through the HIMC, and the types of technologies that are being developed there

Rohit Gupta:Considerations for Biobanking of Clinical Samples

Using the experience of the HIMC Biobank, this presentation will discuss the important considerations for setting up clinical biorepositories, from study design, to quality control, processing protocols, and software integration.

Holden Maecker:Multiplexed Assays for Human Cytokine Detection

Multiple platforms for multiplexed immunoassays exist, of which Luminex is the most widely used. This presentation will focus on the principles of Luminex and MesoScale Discovery platforms, and caveats such as non-linearity, matrix effects, etc. Suggestions for how to report Luminex data will be presented.

Weiqi Wang:Linking T cell receptor repertoire to multi-parametric phenotyping at the single-cell level

As a complement to CyTOF, targeted RNAseq assays can allow exploration of gene expression patterns at the single cell level. For T cells, this can include the sequencing of TCR genes, allowing the determination and tracking of clones, together with their associated gene expression phenotypes.

Alice Fan:Simple Western Nanoimmunoassays (NIA) for Biomarker Studies.

Simple Western Nanoimmunoassay (NIA) is a nanofluidic capillary based ProteinSimple technology that uses isoelectric protein focusing and antibody detection to quantify protein expression and monitor post-translational modifications in limited amounts of starting material. It can measure alterations in post-translational modification in response to targeted therapeutics, and it also allows quantification of protein expression or changes in specific protein isoforms. We have used NIA to analyze proteins in as little as 500 primary cells and have embraced the system to assist in evaluating clinical therapeutics. Thus, NIA may be a promising novel technology for new diagnostic and biomarker studies.

Professor Garry Nolan

Professor, Microbiology & Immunology - Baxter Laboratory

Member, Bio-X Member, Child Health Research Institute Member, Stanford Cancer Institute

Full bio

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