|LAB 1||Build a Flow Cytometer|
|Instructors:||Martin and Wilder|
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.
|LAB 2||Chromosome Analysis|
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.
|LAB 3||Mass Cytometry Laboratory|
|Instructors:||Poulin, Baranov, and Baranov|
Learn more about mass cytometry and how you can implement this exciting technology in your own research. In this laboratory, you will learn the basics of mass cytometry. Introductory topics will include a technology overview, a tour of the Helios mass cytometer, and sample acquisition considerations. We will discuss panel design for mass cytometry including a demonstration of Fluidigm’s Panel Designer tool. We will also cover a roadmap for getting started with your own mass cytometry experiments. Finally, we will go over some data analysis options for high dimensional mass cytometry data including Premium Cytobank and Gemstone.
|LAB 4||Monitoring immune cell functions by mRNA transcription and protein expression|
|Instructors:||Wallace and Soh|
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:
|LAB 5||Advanced Sorting|
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.
|LAB 6||A Typical Cytometry Day|
|Instructors:||Pletcher and Wilshire|
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 tutorial(s):
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 )
|LAB 7||Monitoring Cell Function and Proliferation|
|Instructors:||Tario and Muirhead|
This lab module will consider different methods of measuring immune cell function by flow cytometry including:
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:
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
|LAB 8||Advanced Compensation|
|Instructors:||Konz and Herbert|
In this exciting lab module, we will consider the steps to accurately compensate advanced polychromatic flow cytometry samples. As instrument and reagent vendors are advancing our ability to analyze and sort beyond 16+ antibody/fluorochrome panels, we need to understand the proper methods to compensate our samples and ensure detection of dimly expressed antibodies and unique populations expressed by our cells in our panels. We will understand the effects of fluorescent spillover and the error which can be associated with fluorochrome choice.
This lab will include proper compensation controls to run, acquisition considerations and accurate methods to compensate during and post data acquisition. A plethora of software and instrument platforms will be utilized in this hands-on lab to demonstrate accurate compensation methods.
This is a must take module for those students who are serious about accurate advanced multiparameter compensation.
|LAB 9||Flow Cytometry Analysis of Extracellular Vesicles|
|Instructors:||Rogers and Schmierer|
You have a project to measure extracellular vesicles (EVs), but haven't done it before. You're more than a bit worried, having heard the raging controversies at the regional/national/international meetings about whether anything in the literature on the topic of EVs means anything at all, or even whether a conventional flow cytometer such as yours can even see an EV, let alone measure them quantitatively.
We will look at FCM data from small particles, including EVs, with a critical eye towards assessment of such questions as:
In the hands-on portion of the lab you will have the opportunity to experience the set up and adjustment of the instrument to optimize its ability to detect small particles. Each student will see first-hand the challenges and pitfalls of flow cytometry at the limit.
|LAB 10||Intracellular Cytometry: signaling and cell cycle regulation|
|Instructors:||Hedley, Shankey, and Chow|
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.
|LAB 11||Methods for Apoptosis Detection|
|Instructors:||Telford, Tamul, and Bradford|
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.
|LAB 12||Imaging Flow Cytometry: Combining morphology and classic flow cytometry|
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.
|LAB 13||Automate Cytometry Applications and Visualize High-dimensional Data|
|Instructors:||Hill, Inokuma, and Bagwell|
This laboratory will focus on automating cytometry applications and visualizing high-dimensional data, arguably two of the most important topics in modern cytometry. Cytometry is evolving away from manual and subjective gate-based analysis systems to objective and automated systems. As modern cytometers evolve, the number of correlated measurements is steadily increasing, making it impractical to examine dot-plots. This laboratory will present exciting solutions to both problems using the technique of probability state modeling. The laboratory is entirely hands-on where you will not only get to automate the selection of specific cell types in bone marrow and peripheral blood, but also will visualize and analyze very high-dimensional data.
After completing this laboratory, you will never look at cytometry data in the same way.
|LAB 14||Flow Cytometry Data Acquisition|
|Instructors:||Naivar and Freyer|
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!
|LAB 15||Targeted Biosensors and Drug Discovery|
|Instructors:||Waggoner and Wu|
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.
|LAB 16||Immunofluorescence Analyses|
|Instructors:||Preffer and Kelliher|
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 and complexity/granularity are coupled with this information to identify and describe individual leukocyte cell populations. In our laboratory discussion, we will focus on utilizing a three laser – 10 parameter flow cytometer to primarily characterize human lymphocytes for their expression of commonly assayed immunologic markers.
In these sessions we will describe three commonly used lasers and the related physics that enable the lasers to excite particular fluorochromes. We will integrate this information with the fluorochromes and their characteristics that are detected by the flow cytometer as well as how the instrument isolates and translates each signal into fluorescent channels and ultimate visualization of the resultant data. There will be a major focus on proper instrument set up, quality control using beads as well as cells, compensation theory and staining methods with participants involved in “hands-on” staining and instrument operation. There will be a concentrated discussion about the antibody/antigen reaction, antibody kinetics, how monoclonal antibodies are made, antibody titering, conjugated versus unconjugated antibodies, the importance of choosing the correct clone as well as choosing the correct antibody/fluorochrome combination. We will discuss the mechanics of staining, surface vs. intracellular as well as all the reagents necessary to properly attain successful staining. Finally, we will describe uses of immunophenotyping primarily as it applies to the clinical laboratory, including measuring T, B and NK cell subsets, leukemia and lymphoma diagnostics, quantifying stem cells, monitoring monoclonal antibody therapies, and defining immunodeficiencies. In discussing these topics, we will describe immunophenotypic definitions of these aforementioned cells and related immunobiology. To conclude, we will discuss analysis techniques, software and gating techniques as well as instrumentation, reagent resources and learning tools.
|LAB 17||Cytometric Analysis and Sorting of Complex Tissues and Plants|
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.
|Other Important Locations||Room Number|
|Course office & email||110 (off the atrium)|
|Tissue Culture Hood||236B|
|Tissue Culture Refrigerator||238|
|CO2 Incubator for course use||221|
41st Annual Course in Cytometry, June 16-22, 2018
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Content, instructors, and schedule are subject to change.
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