35th Annual Course in Flow Cytometry
Research Methods and Applications
June 9-15, 2012

Lab Descriptions

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.

Try something new and fun: chromosome analysis and sorting. This is an exciting hands-on lab. You will start with a tissue culture flask containing cells that are ideally suited for this laboratory session. Mitotic cells will be selectively recovered, chromosomes isolated, stained, and analyzed on a flow cytometer. Course participants will be divided into teams of two or three each; each team will prepare and analyze their own chromosome sample. Depending on the instrumentation available, analysis and the critical elements of chromosome analysis and sorting will be covered. Although not every course participant is involved in chromosome analysis, the principles, challenges, and solutions developed in this lab session are common to high-resolution analysis of any sample with multiple subpopulations.  Come see how we resolve 12 populations using a single fluorochrome.

Flow cytometry is a method for analyzing cells for surface and intracellular proteins utilizing one or more lasers and “probes” conjugated to fluorescent tags which can be measured by a photomultiplier tube. In addition, relative measurements regarding cell size and granularity can be coupled with this information to identify and describe individual cell populations. For the immunophenotyping laboratory, we will focus on utilizing 3 laser – 10 parameter flow cytometry to characterize human leukocytes for immunologic markers and some of the physics of light that enable the lasers to excite particular fluorochromes.

We will discuss the three laser system we commonly use in our clinical laboratory as well as our 8-laser research instrument, highlighting the advantages of each platform.. We will discuss the fluorochromes and their characteristics that are detected by a flow cytometer as well as how the instrument isolates and translates each signal into fluorescent channels that translate into the histogram and/or dot plot. We will then discuss proper instrument set up, quality control using beads as well as cells, compensation theory and methods.

Antibodies
We will discuss the antibody/antigen reaction, antibody kinetics, how antibodies are made, antibody titering, conjugated versus unconjugated antibodies, the importance of choosing the correct clone as well as choosing the correct antibody/fluor combinations in multiparameter flow cytometry. Additionally, we will discuss the mechanics of staining, surface vs. intracellular as well as necessary reagents.

Immunophenotyping/Immunology
Next, we will cover the uses of immunophenotyping especially as it applies to the clinical lab including measuring T cell subsets, leukemia and lymphoma characterization, quantifying stem cells, monitoring monoclonal antibody therapies, and defining immunodeficiencies. In discussing these topics, we will define the white blood cells including lymphocyte subsets, platelets and megakaryocytes, red blood cells, and rare cells. We will describe T cells, B cells and NK cells and discuss their development and biology and how it relates to hematologic malignancy diagnosis. For a research perspective, we will also depict how our research lab detects Tregs on the 8-laser platform. Finally, we will discuss analysis techniques, software and gating techniques as well as instrumentation, reagent resources, learning tools and the future of flow cytometry, especially in the clinical lab.

Functional assay for quantitation of oxidative burst - Neutrophils

This laboratory is a hands-on experience in which the participants will perform a kinetic study of the activation of human neutrophils to undergo the oxidative burst pathway. The principles involved and different research applications will be demonstrated and discussed.

Specifically, the participants will examine the time course for neutrophil oxidative activation after stimulation with phorbol myristate acetate (other activators can be used and will be discussed).   The lab will use the oxidation of Dihydrorhodamine123 as the indicator method and the participants will analyze and quantitate results by indexes of mean peak channel fluorescence.  

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 tools 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. Among the topics to be covered will be standard sorting, enrichment sorting, and high speed sorting of various sample types such as, dendritic cells, microglial cells, adherent cells expressing GFP, activated cells, and normal lymphoid cells.

You walk into your laboratory in the morning with plenty of samples to run. What do you do to ensure your instrument is at peak performance? We’ll show you how you QC and run the instrument. We’ll go over the do’s and don’ts for compensation. Everything will be hands on. At the end, we’ll create typical problems on the instruments and let you work as a team to figure them out.

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

• Tetramer staining
• Cytokine production
• Proliferation monitoring using dye dilution
• 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 cells using different types of cell tracking dyes (CFSE, PKH26).
• Staining and analysis of tetramer binding lymphocytes.
• Analysis of 4 color data correlating cytokine expression, tetramer 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
• Quantitative analysis – is my instrument linear across the intensity scale? Does it matter?

Collection and analysis of data on low frequency subpopulations – are these events real or are they junk?

Fluorescent Proteins (FPs) comprise a family of related reporter molecules that can be conveniently expressed and detected within living cells and organisms. In this hands-on laboratory, participants (in teams of two or three) will design detection strategies using various combinations of lasers and optical filters for the analysis and sorting of FP-expressing mammalian cells and bacteria. Team members will install optical filters on the flow cytometers and collect data. Data analysis will reveal the pros and cons of various detection strategies. Due to broad excitation and emission spectra, FPs exhibit significant spectral overlap when used in combination. The principles of fluorescence spectral overlap compensation will be presented. A discussion on the best tactics will follow.

Equipment and materials available for the laboratory:

• Flow cytometers (iCyt): three Eclipse analyzers and one Synergy sorter
• Optical filters encompassing various wavelengths (Chroma Technology)
• WinList 3D analysis software (Verity Software House)
• Fixed mouse cells expressing various combinations of five FPs: ECFP, EGFP, EYFP, DsRed and HcRed
• Fixed bacteria expressing a single FP: LSS-mKate, tdTomato, DsRed2, mStrawberry, mCherry, mKate2, Katushka, mKate, HcRed, mPlum, eqFP650, eqFP670, E2-Crimson or TagRFP657

This laboratory will focus on the principles in building a multicolor flow panel. We will discuss the key factors to keep in mind when designing an 8 color flow panel. Techniques, tools, and tips will be discussed, using a panel of T-cell markers as the model system. Participants will get the chance to design their panel, then "acquire" their panel on a virtual cytometer. Group discussion will include how to use controls to better troubleshoot and optimize panels.

This laboratory will describe the rapidly-growing new flow cytometry application of signal transduction analysis.  These techniques are based on the use of multiple phospho-specific antibodies that recognize activation states of key elements of signal transduction pathways, combined with additional surface phenotypic markers.  This application is particularly useful for studying aberrant signaling pathways in leukemias, and the effects of novel anticancer agents that inhibit these pathways.  We will be working with acute leukemia patient samples as well cell lines.

This laboratory will focus upon different methods for the measurement of apoptosis.  This is a hands-on laboratory where participants will get to perform several techniques as well as participate in the data analysis and group discussion of all the results.  Participants will use both pre-fixed samples as well as fresh cells that have been incubated with or without varying concentrations of an apoptotic inducer.  Approaches will include mitochondrial membrane potential, Annexin V, Caspase 3, FLICA and TUNEL.

Multispectral 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, this technology collects pictures of the cell in brightfield, darkfield and multiple fluorescent channels using the ImageStream or FlowSight (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 staining, running and compensation on a FlowSight. Then we will use data generated on the ImageStreamX to learn 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. 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.

The technique of Probability State Modeling (PSM) eliminates the necessity for gating, accounts for population overlap, and allows users to see all data correlations for any number of parameters with simple-to-understand graphics.  This laboratory will explore the use of PSM to analyze and display multi-parametric cytometry data.  We’ll begin the lab by carefully reviewing the concepts behind this new technique and then we will explore a large number of data sets to see how it works with real data.  Some of the applications will include, but not be limited to, b-cell lineage, neutrophil lineage, and t-cell activation studies.  This is a completely hands-on laboratory.

 

LAB 14	Basic Data Acquisition and Analysis
Location:	Room 24
Instructors:	Novo and Naivar

Description: The laboratory will focus on the key concepts of data acquisition and analysis. Students will acquire data from a simulated flow cytometer and explore the effects of changing key acquisition settings on the instrument. Emphasis will be placed on the key concepts of thresholding, gating, doublet discrimination and coincidence detection. Basic data analysis concepts such as compensation, model fitting, scaling and display parameters will be reviewed with specific examples highlighted the impact they can have on data analysis and interpretation.

Lab developed by Yang Wu, Yehuda Creeger, Larry Sklar, Alan Waggoner and Intellicyt

We will combine a newly-developed biosensor with high-throughput flow cytometry (HTFC) to illustrate the utility of flow cytometry in drug discovery. You will get a chance to learn the standard procedures in the drug discovery field, get familiar with a new type of biosensor, and see how cells respond to drug treatments in front of your eyes. Videos taken in our high-throughput flow cytometry center at the University of New Mexico will also be available to demonstrate the automated processes of the preparation and screening of plates for drug screening purpose.

The participants in the lab will be given a 384-well plate preloaded with 9μL of cells in media including some set as controls to mimic the real process. Several compounds targeting the cell surface receptor will be given to you; all you need to do is add 1 μL of your compound(s) of choice to the non-control well(s) of your choice, and set the plate aside at 37C incubator and wait. Sixty minutes later, we will add 3μL of fluorogens to all the wells and run the plate(s) through HTFC. After which, data will be analyzed, and you can immediately tell whether these compounds will have any effects on the cells. We will reveal the detail about these compounds then. Depending on timing, we may also perform concentration-dependent response with the system as well.

By the end of this course, we hope you will get some idea about the role of high-throughput screening in the field of drug discovery, gain hands-on experience on setting up assays suitable for high-throughput flow cytometry, be familiar with this new type of biosensor, and bring home some thoughts and ideas about what FAP (Fluorescence Activating Proteins) and HTFC can do for your research.

Fisher GW, Adler SA, Fuhrman MH, Waggoner AS, Bruchez MP, Jarvik JW. (2010) Detection and quantification of beta2AR internalization in living cells using FAP-based biosensor technology. J Biomol Screen. 15(6):703-9. Epub 2010 May 20.

Szent-Gyorgyi C, Schmidt BF, Creeger Y, Fisher GW, Zakel KL, Adler S, Fitzpatrick JA, Woolford CA, Yan Q, Vasilev KV, Berget PB, Bruchez MP, Jarvik JW, Waggoner A. (2008) Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nat Biotechnol. 26(2):235-40. Epub 2007 Dec 23.

35th Annual Course in Flow Cytometry,  June 9-15, 2012
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Content, instructors, and schedule are preliminary and subject to change.  
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