Program

Multicolor flow cytometry and panel design track

Total time 4.5 hours (3x90’ practical sessions) plus lectures

Zosia Maciorowski and John Wotherspoon

This Track will cover the tools, steps and procedures needed to design and evaluate multicolor panels, with pitfall and troubleshooting sessions on panel design and advanced compensation problems. The track is targeted to users who will need more than 4-5 color panels for their research and will consist three practical sessions:

Practical 1: Basic multicolor practice

In the practical session, the calculation of stain index will be used to assess fluorochrome brightness and spillover as well as determination of optimal PMT voltage settings and antibody titration. Both automated and manual compensation will be performed, with a discussion of pitfalls and best practices.

Practical 2: Panel Design

This session will first cover the effect of spread due to spillover on the resolution of populations and data quality. The tools necessary to develop a good multicolor panel will be introduced. Antigen density, fluorochrome brightness and spread, antigen co-expression and instrument variation are important aspects to be considered in the decision process.

Practical 3: Panel troubleshooting

In this session, elements of panel and instrument troubleshooting will include discerning the source of the problem, instrument malfunction, instrument settings and how to resolve issues related.

High dimensional data analysis track

Total time 3 hours (2x90’ practical sessions)

Justin Sinnaeve and Sierra Barone

This track is targeted towards intermediate and advanced user, and will introduce users to the principles and applications of high dimensional data analysis, with overviews of the state of the art and tools currently available, and will include practical modules using current software to teach students how to apply these analysis techniques to their own data.

The hands-on modules are designed to introduce high-dimensional data analysis approaches, provide rationale for their use, and implement the computational tools on provided data sets in R. Participants will learn the basics of running code in R and walk through existing data analysis workflow scripts while learning how the different tools can be applied in a variety of contexts. Tools that that will be highlighted in the session include t-SNE, UMAP, FlowSOM, MEM, as well as their use together in the workflow RAPID.

Cell Sorting track

Total time 4.5 hours (3x90’ practical sessions) plus lectures

Geoffrey Osborne, Joe Trotter, Hadas Keren-Shaul

This track will cover advanced applications and the finer aspects of instrument setup, effects of sort conditions on cell viability and functionality, and practical aspects of cloning and index sorting. Factors that affect the efficiency of the sort as well as methods to monitor and maximize the yield and purity of the sort will be detailed and demonstrated. This will inckude techniques for single cell sorting, index sorting, evaluation of recovery for downstream applications and the new and exciting techniques for computational cell sorting.

Practical 1: advanced cell sorting (Geoffrey Osborne)

This wet lab will use different electrostatic cell sorters in practical “hands on” sessions. Rather than teach how to operate a given brand of instrumentation, we aim to stimulate participants to be able to design well thought out sorting experiments based on a solid understanding of the technology involved, leveraging the experimental requirements with realistic expectations. We will demonstrate, and students will be expected to actively participate, in sorting experiments using a variety of cell types.

Practical 2: Single Cell Sorting and downstream Applications (Hadas Keren-Shaul)

Single cell FACS soring enables the isolation of a desired cell type within a population. In this lab module, we will perform single cell sorting and describe potential downstream applications with a focus on single cell RNA-seq. We will also discuss a robust method for massively parallel scRNA-seq that combines gene expression with surface marker levels using indexed FACS sorting.

Practical 3: Computational Cell Sorting (Joe Trotter)

Recent Advances in high-parameter instrumentation and panel design have resulted in the acceptance of dimensionality reduction and clustering techniques in flow cytometry data analysis that produce derived parameters such as locations on a t-SNE map or Cluster ID, providing new perspectives and valuable insights into the data. These computationally derived parameters do not yet exist in any cell sorter. In this module we will introduce a new computational workflow using FlowJo plugins (X-Shift, t-SNE, ClusterExplorer, and HyperFinder) to define training sets for desired populations from which truly optimized gating strategies may be computed and passed to the instrument using only the measurements available on the sorter. In the lab we will discuss approaches, setup the sorter, generate and analyze our multicolor data leveraging clustering and dimensionality reduction techniques, and use software tools to create an optimized gating strategy to sort target cell population of interest.

Single Modules:

Extracellular Vesicles detection

75’

(John Nolan)

Extracellular vesicles (EVs) are small, dim, and difficult to measure by flow cytometry. Conventional flow cytometers designed for lymphocyte analysis lack the sensitivity to measure EVs well, and the literature is rife with artifacts that result from this. The use of proper experimental design, including appropriate controls and calibration can help identify these artifacts and enable reporting of results in a manner that can allow comparison between labs and among instruments. The ISAC-ISEV-ISTH EV FFC Working Group, a collaborative group of more than a dozen labs using FC to analyze EVs, has developed a framework to guide the reporting of EV FC analysis methods and results to facilitate standardization and progress in the field. This lab module will illustrate some of the key issues of experimental design and measurement calibration for EV FC.

Advanced methods - Cell Signaling

(David Hedley and Sue Chow)

90’

The lab covers methodology for intracellular antigen measurement by flow cytometry, with emphasis on the dynamics of signaling pathways. We illustrate this by tracking the early signaling events in LPS-activated peripheral blood monocytes, then following through to the activation of the NFkB transcription factor, and production of the inflammatory cytokines IL-6 and IL-1. We will take you through the practical steps to produce high quality data using whole blood samples, combining a wide range of intracellular targets while preserving surface immunophenotype and light scatter signals.

Imaging Flow Cytometry

(Ziv Porat)

90’

Imaging Flow Cytometry combines the high-throughput quantification of flow cytometry with the information rich imagery of microscopy. This allows novel applications for flow cytometry as protein co-localization, cell-cell interaction, cellular morphology etc. The module will review the principles of Imaging Flow cytometry and introduce the concepts and principles of data analysis followed by example applications for this technology. The practical module will consist of data acquisition and analysis on the Imagestream for a number of different applications, including NFkB nuclear translocation and apoptosis.