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Lab Descriptions

Labs and instructors from the 2018 Annual Course in Cytometry

This lab is designed to give the students a better appreciation of the inner workings of a flow cytometer, taking away some of the mysteries of what is hidden inside the cabinet. In this lab session you will assemble a small flow cytometer and use it to measure a sample of fluorescent microspheres. You will learn some of the important steps used in setting up a flow cytometer, some diagnostic clues that are useful for evaluating a flow cytometer's performance and an appreciation for what is involved in constructing flow cytometers.

This compact flow cytometer is assembled using modular parts according to a detailed protocol. It includes the following components: laser, laser beam shutter, laser power attenuator, beam block, CCD camera and video monitor (used for viewing the laser beam - sample stream intersection region), flow chamber, fluorescence collection optics, fluorescence detector, electronics, oscilloscope and a computer.

Because you will be installing and aligning optical components along a laser beam line good laser safety practices will be discussed and stressed. By the end of the lab session you will have assembled a working cytometer and will be analyzing the microsphere sample and optimizing the final adjustments to obtain the best CV. In recent years, CVs below 2% have been achieved.

Flow cytometry is ideally suited for measurement of single cell suspensions, and the standard designs of flow cytometers and cell sorters are based on typical size ranges of blood cells, with diameters <20 microns. However, many eukaryotic cells are larger than this, sometimes much larger. Eukaryotic organisms are also infrequently found in the form of natural single cell suspensions, instead being found as tissues and organs, which are complex interspersions of different cell types. Converting organs and tissues to single cell suspensions is not necessarily a simple task, since it requires dissolution of the extracellular matrices that interlink the cells. This laboratory provides practical strategies for handling unusual samples in flow cytometry and cell sorting, including dealing with large cells and other biological particles, and with non-mammalian species. It also demonstrates how simple homogenization techniques can be used to release suspensions of nuclei for flow analysis and sorting. All flow operators should benefit from the materials covered in this lab, since inevitably they will be confronted by users that wish to analyze and sort unusual samples.

New platforms allow for cytometry to be performed with 40+ parameters, bringing new opportunities and challenges. This lab will provide an overview of the benefits of different approaches in high dimensional instrumentation, including mass cytometry, spectral instruments and others. The challenges associated with these instruments and approaches to compensation and unmixing will be covered. Students will stain PBMC with a high-dimensional panel, then rotate through several different instruments for hands-on exposure to the workflow for each one. They will also practice advanced analysis methods (e.g., viSNE) for high-dimensional data using Cytobank.

The identification of distinct T helper lymphocyte (Th1/2/17) and monocyte (M1/2) subsets with polarized cytokine production has opened up new fields in immunobiology. Of the several alternative methods of monitoring cytokine production, flow cytometric analysis of intracellular staining has distinct advantages and pitfalls. It allows high throughput of samples and multiparameter characterisation of cytokine production on a single cell basis without the need for prolonged in vitro culture and cloning. However, these methods can cause changes in cell surface phenotype which can make interpretation difficult. We will together explore methods to measure cytokines and monokines by both protein and mRNA methods. This lab will:

• Follow the kinetics of antigen binding, NFkB translocation, mRNA transcription, and protein expression
• Setup and experimentally measure TH1/2/17 cytokines in activated T cells by flow cytometry
• Discuss the methodological considerations to perform cytokine mRNA and protein measurements
• Correlate the transcription of mRNA with protein expression in LPS activated monocytes
• Correlate cytokine production with chemokine expression patterns

This practical laboratory session will focus on several areas of interest in cell sorting that apply to particle sorting in general. We will cover instrument setup based on the task at hand. In other words, how to realistically approach optimizing nozzle size, stream stability, deflection envelope, break off, drop rate and sample rate for any given experiment. The lab will try to provide the attendee with approaches for use in their own facility in problem solving a wide variety of sorting experiments, regardless of the cytometer they use, including suggestions on advising facility users on sample preparation.

Traditional flow cytometry has for decades embraced analyzing and sorting cell populations using subjective classification approaches based on Boolean combinations of user drawn geometrical regions over one- or two-dimensional data projections. These gating strategies often evolve from a priori knowledge and assumptions of the investigator. As a result, manually generated gates, while helpful to explore and reveal some underlying biology, may be biased and suboptimal to efficiently enrich the phenotypes to be sorted. With the advent of high-parameter cell sorters and computational analysis, the challenges deepen if phenotypes to be sorted are classified by clustering routines, identified by machine learning approaches, or rendered by dimensionality reduction techniques such as tSNE or UMAP. We will use computational tools to classify and sort using objectively efficient strategies.

You walk into your laboratory in the morning with plenty of samples to run. What do you do to ensure your experiment will work at each step from sample prep -> acquisition -> sorting your cells? We will take a "Forensic Cytometry" approach to your flow experiment by reviewing what can go wrong... and show you how to prevent it by following the 9 Practical Rules of Flow Cytometry.

We'll review how to prevent bad data during sample prep by titering your antibodies, Fc blocking, adding a viability dye, and doublet removal. On the machine we'll go over QC and how to set voltages. We'll take apart the analyzer so that you can see which components are being adjusted and not view the machine as a "mysterious black box". We will go over the do's and don'ts for compensation as well as FMO controls for gating. We'll then move to the sorter where we will guide you through alignment of lasers, stream and pinholes - we guarantee that anyone can do it!

If you are somewhat new to flow cytometry, we recommend you prepare for this course by viewing the following tutorials:

Introduction to Flow Cytometry (http://media.invitrogen.com.edgesuite.net/tutorials/4Intro_Flow/player.html)
Flow Cytometry Data Analysis (http://media.invitrogen.com.edgesuite.net/tutorials/5Data_Analysis/player.html )

This lab module will consider different methods of measuring immune cell function by flow cytometry including:

• Multimer staining of antigen specific T cells
• Proliferation monitoring using dye dilution
• Cytokine production in leukocyte subsets
• Phagocytosis and cytotoxicity assays

The focus of the laboratory will be critical issues for cell function assays using commercially available probes. Students will be divided into small groups for hands-on experience with:

• Staining and analysis of multimer binding lymphocytes.
• Staining and proliferation analysis using protein-reactive or membrane intercalating dyes (CFSE, PKH26 and newer analogs of each).
• Probe selection and analysis of multicolor data correlating cytokine expression, multimer binding and differential lymphocyte subset proliferation.

We will also cover several instrument setup and data collection issues likely to be of interest even if your laboratory is not already doing functional assays. These will include

• Color compensation - recognizing problems, optimizing probe combinations to minimize them
• Collection and analysis of data on low frequency subpopulations - are these events real or are they junk?

In this lab students will walk through the workflow of designing an optimized, rigorous, and reproducible flow cytometry panel. The tips and tricks shared in this lab will be applicable to all panels, small or large. With the goal of maximizing sensitivity for each marker detected in rigorous and reproducible assay, we'll discuss the following:

• Power calculations to determine sample size
• Sample preparation
• Protocol Development & Optimization
• Annotation & Record Keeping
• Characteristics of available fluorochromes
• Characteristics of markers of interest
• How to optimally pair markers with fluorochromes
• Proper Controls
• Instrument characteristics
• Spillover spreading
• Assay Standardization
• Considerations for High Dimensional Data Analysis

Students attending this lab should walk away with an understanding of proper considerations and a workflow for designing an optimal, rigorous, and reproducible flow cytometry assay.

Flow Cytometry is the most commonly used technique to characterize extracellular vesicles (EVs). However, most flow cytometers were designed for the analysis of whole cells. Therefore, traditional approaches to instrument set up and acquisition are inadequate for the measurement of sub-micron particles.

The first step in the characterization of EVs by flow cytometry is characterizing the instrument's sensitivity for scatter and fluorescence and understanding the measurement limitations and special considerations for particles < 0.5u. In this lab we will demonstrate best practices for measuring EVs (and other small particles), including scatter and fluorescence calibration, approach to establishing instrument settings, and the necessary controls for the interpretation of the data obtained. We will use well-characterized reference materials to achieve these goals, as well as two different instrument platforms: the Aurora spectral cytometer and the ImagestreamX imaging flow cytometer.

Much of the regulation and coordination of cell function is controlled by post-translational modifications (phosphorylation, methylation, acetylation, glycosylation, ubiquination, etc), predominantly of intracellular or intranuclear proteins. Specific modifications regulate fundamental cell processes (death, division, differentiation), and detection or quantification of specific protein modifications can be uniquely monitored in complex or heterogeneous cell mixtures using flow cytometry.

This lab covers the technical aspects of intracellular antigen staining for flow cytometry. We will present and discuss in detail a Toolbox, including a basic approach to fixation and permeabilization to provide access to cytoplasmic and nuclear compartments, variations to allow simultaneous detection of cell surface epitopes, and a technique to "unmask" otherwise difficult to detect cellular of nuclear epitopes. In the lab, we will illustrate practical applications, including, (1) the activation of signal transduction pathways that regulate the acute inflammatory response via the NFkB transcription factor, and (2) molecular mechanisms that regulate movement through the cell division cycle.

Flow cytometry is a method for analyzing cells for multiple surface and intracellular proteins utilizing excitation lasers and monoclonal antibodies conjugated to unique fluorescent tags. Additionally, simultaneous light scatter measurements that impart cell size (forward light scatter) and complexity (90° light scatter) are coupled with this information to identify and describe individual leukocyte cell populations.

In our laboratory, we will go through the steps of basic panel design utilizing common T-cell lineage and subset markers. We will perform antibody titration and calculate the antibody's stain index and compare to signal to noise ratio to identify the optimal titration for our experiment. Four different panels using the results of the titration are prepared and evaluated to determine the optimal antibody clone and the optimal fluorochrome. The titrations and panel preparation will be prepared by lab participants and acquired on a 10-color BD FACSLyric flow cytometer.

Throughout the lab, we will discuss the mechanics of staining, the antibody-antigen reaction, antibody kinetics, antibody titration, and the importance of choosing the optimal monoclonal antibody clone and pairing it with a fluorochrome that maximizes signal resolution.

Imaging flow cytometry resembles classic flow cytometry in that fluorescent data are collected on single cells in flow. However instead of total cellular intensity values quantitated on photomultiplier tubes, CCD cameras collect the emissions as pictures of the cell in brightfield, darkfield and multiple fluorescent channels using the ImageStream (Amnis Corp). The images can then be analyzed for levels of intensity as well a number of morphological aspects of the intensity (shape, size, texture) as well as comparing locations of different stains (e.g. nuclear and transcription factor stains).

However, with great power comes great complexity and the analysis can be daunting! During the lab, we learn the basics of running samples on the ImageStream or Flowcyte and how that differs from standard flow. The bulk of the time will be spent analyzing data learning how to translate characteristics of size, shape and texture we see with our own eyes in the images into quantifiable features in the analysis software. We will also learn how to select the best feature for the best discrimination. We will have training samples for new users of this technology as well more advanced training on the power of masking and complex analysis. The goal of the course is to enable the participants to know if you can visually observe a difference in cells, you can use flow cytometry to quantitate it. Please bring your computers if you have them because you can do your analysis on them. We will also have computers for those who don't have them.

Recent advancements in cytometry allow us to measure an increasing number of features per cell; generating huge high-dimensional datasets. This creates challenges for data analysis. Traditional approaches based on subjective manual gating on biaxial plots are not suitable. New computational techniques are being developed to analyze, visualize and interpret these data. Non-linear dimensionality reduction techniques, automated analysis, normalization and batch effect removal, automated quality assessments, cell and sample classification and other computational approaches are gaining on importance.

This lab will introduce attendees to some of these approaches. We will discuss the pros and cons and practical aspects of different methods using a hands-on approach with a high-dimensional dataset. You will learn what PCA, t-SNE, UMAP and Cen-se' can do for you and how they differ. We will develop an analysis strategy using probability state modeling and GemStone™ 2.0. We will teach you how to perform and interpret dimensionality reduction, automated gating and other computational analysis approaches in FlowJo™. Finally, attendees will perform analyses with R-based and other algorithmic approaches, while discussing the necessary theoretical background of doing so, including fundamentals of computational biology and statistical methodology used in those assays.

This laboratory will primarily focus on the key concepts of data acquisition, and also cover some basic data analysis. Regardless of whether you are going to gate or model your data to get your final result, and regardless of which instrument you are using, it is important to acquire flow data as accurately as possible. It is much better to collect your data properly up front, rather than try to "fix" poor data after the fact (many acquisition problems simply cannot be "fixed").

Small teams of students will use flow cytometer simulators to explore critical factors that affect data acquisition (sample preparation, thresholding, PMT voltage, coincidence, aggregates, sample throughput). Students will be able to reconfigure the cytometer simulator in ways that are impossible for real instruments in order to provide a more intuitive feel for how different conditions can affect data acquisition. We will also cover the basics of data visualization and gating, which are important for evaluating and adjusting acquisition parameters.

Finally, the simulator will be used to explore some basic examples of compensation to give students a much better feel for when compensation is important, why it is needed, and how it improves the data that are collected. Because we will be using a simulator, no cells will be harmed and no one will run out of sample! No lab coats, gloves or goggles required!

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