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Flow Cytometry
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BD Biosciences 2350 Qume Drive San Jose, CA 95131- 1-800-448-
Manual Part Number: 11-11032- April, 2000
Introduction to Flow Cytometry: A Learning Guide
Copyright
© 2000 Becton, Dickinson and Company. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, stored in retrieval systems, or translated into any language or computer language, in any form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without the prior written permission of BD Biosciences, 2350 Qume Drive, San Jose, CA 95131, United States of America.
Disclaimer
BD Biosciences reserves the right to change its products and services at any time to incorporate the latest technological developments. This guide is subject to change without notice. BD Biosciences welcomes customer input on corrections and suggestions for improvement. Although this guide has been prepared with every precaution to ensure accuracy, BD Biosciences assumes no liability for any errors or omissions, nor for any damages resulting from the application or use of this information.
Trademarks
Apple, the Apple logo, Mac, Macintosh, and Power Macintosh are trademarks of Apple Computer, Inc., registered in the U.S. and other countries. Finder is a trademark of Apple Computer, Inc. Modfit LT and QuantiCALC are trademarks of Verity Software House, Inc. C ell Quest, FACS, FACSCalibur, FACScan, FACSort, FACStarPLUS^ , and FACS Vantage are trademarks of Becton, Dickinson and Company.
Introduction to Flow Cytometry: A Learning Guide
ii
7.1 How Lasers Work...................................................... 43 7.2 Laser Alignment....................................................... 44
Chapter 8: Answer Key 47
iii
Preface
Learning to operate a flow cytometer is best achieved by using the instrument. However, understanding the principles underlying this technology greatly facilitates the process.
This document contains basic information on flow cytometry. Differences between flow cell–based benchtop cytometers (FACScan™, FACSort™, FACSCalibur™, and BD LSR) and stream-in-air cytometers (FACS Vantage™, FACSVantage™ SE, and FACStarPLUS™^ ) are described in relevant sections. Reading this material and answering the questions at the end of each section will enhance your hands-on training experience during Operator Training at BD Biosciences. This assignment will take approximately 2.5 hours to complete. Please review it before you attend the training session. An answer key is provided. If you have any questions or problems in the US, call 1-800-448-2347, Option 4. In Europe, contact your local application specialist.
For more information on general flow cytometry, review the following:
Flow cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. The properties measured include a particle’s relative size, relative granularity or internal complexity, and relative fluorescence intensity. These characteristics are determined using an optical-to-electronic coupling system that records how the cell or particle scatters incident laser light and emits fluorescence.
A flow cytometer is made up of three main systems: fluidics, optics, and electronics.
In the flow cytometer, particles are carried to the laser intercept in a fluid stream. Any suspended particle or cell from 0.2–150 micrometers in size is suitable for analysis. Cells from solid tissue must be disaggregated before analysis. The portion of the fluid stream where particles are located is called the sample core. When particles pass through the laser intercept, they scatter laser light. Any fluorescent molecules present
Introduction to Flow Cytometry: A Learning Guide
on the particle fluoresce. The scattered and fluorescent light is collected by appropriately positioned lenses. A combination of beam splitters and filters steers the scattered and fluorescent light to the appropriate detectors. The detectors produce electronic signals proportional to the optical signals striking them.
List mode data are collected on each particle or event. The characteristics or parameters of each event are based on its light scattering and fluorescent properties. The data are collected and stored in the computer. This data can be analyzed to provide information about subpopulations within the sample (Figure 1-1).
Figure 1-1 Scattered and emitted light signals are converted to electronic pulses that can be processed by the computer
laser
sample core
data displays
electronic pulses
Introduction to Flow Cytometry: A Learning Guide
The purpose of the fluidics system is to transport particles in a fluid stream to the laser beam for interrogation. For optimal illumination, the stream transporting the particles should be positioned in the center of the laser beam. In addition, only one cell or particle should move through the laser beam at a given moment.
To accomplish this, the sample is injected into a stream of sheath fluid within the flow chamber. The flow chamber in a benchtop cytometer is called a flow cell and the flow chamber in a stream-in-air cytometer is called a nozzle tip. The design of the flow chamber causes the sample core to be focused in the center of the sheath fluid where the laser beam will then interact with the particles.
Based on principles relating to laminar flow, the sample core remains separate but coaxial within the sheath fluid. The flow of sheath fluid accelerates the particles and restricts them to the center of the sample core. This process is known as hydrodynamic focusing. For an illustration of hydrodynamic focusing in each type of flow cell, see Figure 2-1 and Figure 2-2.
Increasing the sample pressure increases the flow rate by increasing the width of the sample core. This, in turn, allows more cells to enter the stream within a given moment. With a wider sample core, some cells could pass through the laser beam off-center and intercept the laser beam at a less optimal angle. However, this might be appropriate for your application.
Proper operation of fluidic components is critical for particles to properly intercept the laser beam. Therefore, the operator must always ensure that the fluidics system is free of air bubbles and debris and is properly pressurized at all times.
Introduction to Flow Cytometry: A Learning Guide
1 The purpose of the fluidics system in a flow cytometer is:
2 What two factors can affect illumination of the particles within the laser beam?
3 How many cells or particles should pass through the laser beam at a given time?
4 The particle suspension is injected into _________________ within the _________________.
5 The process of centering the sample core within the sheath fluid is known as:
6 Which regulator controls the diameter of the sample core?
7 What are the three possible pressure settings for a benchtop flow cytometer?
8 Increasing sample pressure ______________ the sample flow rate and the ________________ of the sample core.
9 Good data resolution is required for DNA studies. What flow rate is recommended?
10 A wider sample core decreases resolution.
T F
11 A high flow rate can be used when performing qualitative measurements.
T F
Introduction to Flow Cytometry: A Learning Guide
Side-scattered light (SSC) is proportional to cell granularity or internal complexity. SSC is a measurement of mostly refracted and reflected light that occurs at any interface within the cell where there is a change in refractive index (Figure 3-1). SSC is collected at approximately 90 degrees to the laser beam by a collection lens and then redirected by a beam splitter to the appropriate detector.
Figure 3-1 Light-scattering properties of a cell
Correlated measurements of FSC and SSC can allow for differentiation of cell types in a heterogeneous cell population. Major leucocyte subpopulations can be differentiated using FSC and SSC (Figure 3-2).
Figure 3-2 Cell subpopulations based on FSC vs SSC
light source
side scatter detector
forward scatter detector
neutrophils
monocytes
lymphocytes
lysed whole blood
Chapter 3: Generation of Scatter and Fluorescence
1 When does light scattering occur?
2 Which key cell components contribute to light scatter?
3 Light scattered in the same direction as the laser beam is called:
4 FSC is proportional to: ____________________________________________
5 Light scatter collected at 90 degrees to the laser beam is called:
6 SSC is proportional to the ___________ or _____________ of the cell.
7 Correlated measurements of both _______________ and _____________ can
allow differentiation of cells types in a heterogeneous cell population.
Figure 3-3 Absorption spectra of four common fluorochromes
Figure 3-4 Emission spectra of the fluorochromes shown in Figure 3-
When a fluorescent dye is conjugated to a monoclonal antibody, it can be used to identify a particular cell type based on the individual antigenic surface markers of the cell (Figure 3-5 on page 18). In a mixed population of cells, different fluorochromes can be used to distinguish separate subpopulations. The staining pattern of each subpopulation, combined with FSC and SSC data, can be used to identify which cells are present in a sample and to count their relative percentages. The cells can also be sorted if desired.
FITC = fluorescein isothiocyanate PE = phycoerythrin PerCP = peridinin chlorophyll protein APC = allophycocyanin
Introduction to Flow Cytometry: A Learning Guide
Figure 3-5 Specific binding of fluorochrome-labeled antibodies to cell surface antigens
1 When fluorescent compounds absorb light energy and then release excess energy, they emit ____________________.
2 Characteristic wavelength ranges at which fluorescent compounds can be excited are called _________________________________________.
3 The longer wavelengths of light emitted by a fluorochrome make up its _______________________.
4 Which laser is most commonly used in flow cytometry? _________________
5 The FITC and PE fluorochromes are excited by this emission wavelength of an argon-ion laser: _________________
6 Two fluorescent dyes commonly used in flow cytometry are ___________ and ____________.
7 Fluorochrome-labeled antibodies are used to detect ______________________.
fluorochrome-labeled antibodies
antigenic surface marker
