33rd Annual Course in Flow Cytometry
Research Methods and Applications
June 19-25, 2010

Lab Descriptions

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 making final adjustments to obtain the best CV for the week. In years past CVs below 1% 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.

This laboratory will describe the procedures for immunophenotyping by multiparameter flow cytometry.

Objectives:

1. We will review the kinetics of antibody binding to cells. They are the tools of the process.

2. Methods for properly titering antibodies and combining them for 2 – 4 color analysis will be discussed.

3. The method for intra and extra cellular staining using the indirect and direct methods will be described and demonstrated.

Questions and personal experiences of participants are most welcome.

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.

In order to study the cell cycle by flow cytometry, we must understand how to make a flow cytometer provide precise results. Therefore this laboratory module has two goals: teaching how to do cell cycle analysis and also teaching the principles of flow cytometry as they affect all forms of flow analysis. We will start by talking about the ways that cells can be stained for DNA content; we will then look at the ways that the use of a flow cytometer can affect the data derived from those stained cells; and finally we will discuss the ways that analysis of the data can affect our conclusions.

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. This laboratory will perform:

• Flow cytometer setup
• Analysis of fixed Sp2/0 mouse hybridoma cells:
• Cells expressing ECFP, EGFP, EYFP, DsRed or HcRed
• Cells expressing four FPs (ECFP/EGFP/EYFP/DsRed)
• Cells expressing five FPs (ECFP/EGFP/EYFP/DsRed/HcRed)
• Software compensation: hands-on practice by class participants
• Sorting of fixed Sp2/0 cells: hands-on practice by class participants
• Simultaneous detection of GFP and DNA content in plant nuclei4. Sorting of fixed Sp2/0 cells: hands-on practice by class participants

This laboratory will be centered on multi-variate cell cycle analysis. The objectives are to examine two backbone analyses. The first is centered on mitosis and we will look at samples stained for DNA content, cyclin A, cyclin B1, and phospho-S10-histone H3. This analysis identifies cell biochemical states that are associated with prophase, prometaphase, metaphase, late mitosis, and cytokinesis. The second is aimed at gaining information about G1 and will include samples stained for DNA content, phospho-S10-histone H3, and DNA-bound mcm-6 and DNA-bound PCNA. This allows detection of uncommitted and committed G1 cells, S, and G2 cells. The two assays are incompatible and cannot be physically integrated because they each employ mutually exclusive fixation/permeabilization front ends.

In the lab, we will discuss fixation/permeabilization briefly, and a chemically rational staining protocol that has worked for us for many years. Then we will replay list mode data to illustrated instrumental setup. We plan to do this for FACS Diva as we would setup to acquire data on a 4 laser LSR II flow cytometer (BD Biosciences). Additionally, we will replay data obtained on a CompuCyte laser scanning cytometer to illustrate the process for both flow and laser scanning cytometry. Lastly, we will attempt to integrate the two assays analytically with Gemstone to create a comprehensive multivariate analysis. If we have time, we will discuss BrdU labeling and cell kinetics analysis.

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 is a new technology utilizing the ImageStream (Amnis Corp.) that captures brightfield, darkfield and multiple fluorescent images of individual cells in flow.  The images can then be analyzed for levels of fluorescence intensity as well as the shape and size of the area of fluorescence.

However, with great power comes great complexity and the analysis can be daunting!  During the lab, we will stain and run samples, and then compensate the files. The bulk of the analysis time will be spent 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.  As our model system we will be analyzing bone marrow erythroblasts and granulocytes, combining cell surface immunophenotype, nuclear morphology and cell shape characteristics to delineate maturational series.  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.

33rd Annual Course in Flow Cytometry,  June 19-25, 2010
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