Flow Cytometry Protocols 2d ed - Teresa S. Hawley.pdf - Biologia komórki i molekularna - carambola

METHODS IN MOLECULAR BIOLOGY TM

Volume 263

Flow Cytometry

Protocols

S ECOND E DITION

Edited by

Teresa S. Hawley

Robert G. Hawley

Teresa S. Hawley

Robert G. Hawley

METHODS IN MOLECULAR BIOLOGY TM

Flow Cytometry

Protocols

S ECOND E DITION

Edited by

Flow Cytometry

An Introduction

Alice L. Givan

Summary

A flow cytometer is an instrument that illuminates cells (or other particles) as they flow indi-

vidually in front of a light source and then detects and correlates the signals from those cells that

result from the illumination. In this chapter, each of the aspects of that definition will be

described: the characteristics of cells suitable for flow cytometry, methods to illuminate cells, the

use of fluidics to guide the cells individually past the illuminating beam, the types of signals

emitted by the cells and the detection of those signals, the conversion of light signals to digital

data, and the use of computers to correlate and analyze the data after they are stored in a data file.

The final section of the chapter will discuss the use of a flow cytometer to sort cells. This chap-

ter can be read as a brief, self-contained survey. It can also be read as a gateway with signposts

into the field. Other chapters in this book will provide more details, more references, and even

some controversy about specific topics.

Key Words

Flow cytometry, fluidics, fluorescence, laser.

1. Introduction

An introductory chapter on flow cytometry must first confront the difficulty

of defining a flow cytometer. The instrument described by Andrew Moldavan in

1934 (1) is generally acknowledged to be an early prototype. Although it may

never have been built, in design it looked like a microscope but provided a cap-

illary tube on the stage so that cells could be individually illuminated as they

flowed in single file in front of the light emitted through the objective. The

signals coming from the cells could then be analyzed by a photodetector

attached in the position of the microscope eyepiece. Following work by Coul-

From: Methods in Molecular Biology: Flow Cytometry Protocols, 2nd ed.

Edited by: T. S. Hawley and R. G. Hawley © Humana Press Inc., Totowa, NJ

Givan

ter and others in the next decades to develop instruments to count particles in

suspension ( see refs. 2–5) ,adesign was implemented by Kamentsky and

Melamed in 1965 and 1967 (6 , 7) to produce a microscope-based flow cytome-

ter for detecting light signals distinguishing the abnormal cells in a cervical

sample. In the years after publication of the Kamentsky papers, work by

Fulwyler, Dittrich and Göhde, Van Dilla, and Herzenberg ( see refs. 8–11) led to

significant changes in overall design, resulting in a cytometer that was largely

similar to today’s cytometers. Like today’s cytometers, a flow cytometer in 1969

did not resemble a microscope in any way but was still based on Moldavan’s

prototype and on the Kamentsky instrument in that it illuminated cells as they

progressed in single file in front of a beam of light and it used photodetectors to

detect the signals that came from the cells ( see Shapiro [12] and Melamed

[13 , 14] for more complete discussions of this historical development). Even

today, our definition of a flow cytometer involves an instrument that illuminates

cells as they flow individually in front of a light source and then detects and cor-

relates the signals from those cells that result from the illumination.

In this chapter, each of the aspects in that definition are described: the cells,

methods to illuminate the cells, the use of fluidics to make sure that the cells

flow individually past the illuminating beam, the use of detectors to mea-

sure the signals coming from the cells, and the use of computers to correlate the

signals after they are stored in data files. As an introduction, this chapter can

be read as a brief survey; it can also be read as a gateway with signposts into

the field. Other chapters in this book (and in other books [e.g., refs. 12 , 15–24)

provide more details, more references, and even some controversy concerning

specific topics.

2. Cells (or Particles or Events)

Before discussing “cells,” we need to qualify even that basic word. “Cytome-

ter” is derived from two Greek words, “

κντοζ

”, meaning

measure. Cytometers today, however, often measure things other than cells. “Par-

ticle” can be used as a more general term for any of the objects flowing through

a flow cytometer. “Event” is a term that is used to indicate anything that has

been interpreted by the instrument, correctly or incorrectly, to be a single parti-

cle. There are subtleties here; for example, if the cytometer is not quick enough,

two particles close together may actually be detected as one event. Because

most of the particles sent through cytometers and detected as events are, in fact,

single cells, those words are used here somewhat interchangeably.

Because flow cytometry is a technique for the analysis of individual parti-

cles, a flow cytometrist must begin by obtaining a suspension of particles. His-

torically, the particles analyzed by flow cytometry were often cells from the

µετρον

”, meaning container, receptacle,

or body (taken in modern formations to mean cell), and “

Flow Cytometry: An Introduction

blood; these are ideally suited for this technique because they exist as single

cells and require no manipulation before cytometric analysis. Cultured cells or

cell lines have also been suitable, although adherent cells require some treat-

ment to remove them from the surface on which they are grown. More recently,

bacteria (25 , 26) ,sperm (27 , 28) , and plankton (29) have been analyzed. Flow

techniques have also been used to analyze individual particles that are not cells

at all (e.g., viruses [30] , nuclei [31] , chromosomes [32] ,DNA fragments [33] ,

and latex beads [34] ). In addition, cells that do not occur as single particles

can be made suitable for flow cytometric analysis by the use of mechanical

disruption or enzymatic digestion; tissues can be disaggregated into individual

cells and these can be run through a flow cytometer. The advantage of a method

that analyzes single cells is that cells can be scanned at a rapid rate (500 to

>5000 per second) and the individual characteristics of a large number of cells

can be enumerated, correlated, and summarized. The disadvantage of a single-

cell technique is that cells that do not occur as individual particles will need to

be disaggregated; when tissues are disaggregated for analysis, some of the char-

acteristics of the individual cells can be altered and all information about tissue

architecture and cell distribution is lost.

In flow cytometry, because particles flow in a narrow stream in front of a

narrow beam of light, there are size restrictions. In general, cells or particles

must fall between approx 1

m and approx 30

10 6 /mL. In this suspension, they will flow through the

cytometer mostly one by one. The light emitted from each particle will be

detected and stored in a data file for subsequent analysis. In terms of the emit-

ted light, particles will scatter light and this scattered light can be detected.

Some of the emitted light is not scattered light, but is fluorescence. Many par-

ticles (notably phytoplankton) have natural background (auto-) fluorescence

and this can be detected by the cytometer. In most cases, particles without

intrinsically interesting autofluorescence will have been stained with fluorescent

dyes during preparation to make nonfluorescent compounds “visible” to the

cytometer. A fluorescent dye is one that absorbs light of certain specific colors

and then emits light of a different color (usually of a longer wavelength). The

fluorescent dyes may be conjugated to antibodies and, in this case, the fluo-

rescence of a cell will be a readout for the amount of protein/antigen (on the

cell surface or in the cytoplasm or nucleus) to which the antibody has bound.

10 5

to 5

m in diameter. Special cytome-

ters may have the increased sensitivity to handle smaller particles (such as DNA

fragments [33] or small bacteria [35] ) or may have the generous fluidics to

handle larger particles (such as plant cells [36] ). But ordinary cytometers will,

on the one hand, not be sensitive enough to detect signals from very small par-

ticles and will, on the other hand, become obstructed with very large particles.

Particles for flow cytometry should be suspended in buffer at a concentration

of about 5

Givan

Some fluorochrome-conjugated molecules can be used to indicate apoptosis

(37) . Alternatively, the dye itself may fluoresce when it is bound to a cellular

component. Staining with DNA-sensitive fluorochromes can be used, for exam-

ple, to look at multiploidy in mixtures of malignant and normal cells (31) ; in

conjunction with mathematical algorithms, to study the proportion of cells in

different stages of the cell cycle (38) ; and in restriction-enzyme-digested mate-

rial, to type bacteria according to the size of their fragmented DNA (39) . There

are other fluorochromes that fluoresce differently in relation to the concentra-

tion of calcium ions (40) or protons (41 , 42) in the cytoplasm or to the poten-

tial gradient across a cell or organelle membrane (43) . In these cases, the

fluorescence of the cell may indicate the response of that cell to stimulation.

Other dyes can be used to stain cells in such a way that the dye is partitioned

between daughter cells on cell division; the fluorescence intensity of the cells

will reveal the number of divisions that have occurred (44) . Chapters in this

book provide detailed information about fluorochromes and their use. In addi-

tion, the Molecular Probes (Eugene, OR) handbook by Richard Haugland is

an excellent, if occasionally overwhelming, source of information about fluor-

escent molecules.

The important thing to know about the use of fluorescent dyes for staining

cells is that the dyes themselves need to be appropriate to the cytometer. This

requires knowledge of the wavelength of the illuminating light, knowledge of

the wavelength specificities of the filters in front of the instrument’s photode-

tectors, and knowledge of the absorption and emission characteristics of the

dyes themselves. The fluorochromes used to stain cells must be able to absorb

the particular wavelength of the illuminating light and the detectors must have

appropriate filters to detect the fluorescence emitted. For the purposes of this

introductory chapter, we now assume that we have particles that are individu-

ally suspended in medium at a concentration of about 1 million/mL and that

they have been stained with (or naturally contain) fluorescent molecules with

appropriate wavelength characteristics.

3. Illumination

In most flow cytometers, fluorescent cells are illuminated with the light from

a laser. Lasers are useful because they provide intense light in a narrow beam.

Particles in a stream of fluid can move through this light beam rapidly; under

ideal circumstances, only one particle will be illuminated at a time, and the

illumination is bright enough to produce scattered light or fluorescence of

detectable intensity.

Lasers in today’s cytometers are either gas lasers (e.g., argon ion lasers or

helium–neon lasers) or solid-state lasers (e.g., red or green diode lasers or the

relatively new blue and violet lasers). In all cases, light of specific wavelengths

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