Givan A.L. Flow Cytometry. First Principles

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that many benchtop ¯ow cytometers have ``parameters to spare,''

current clinical protocols for lymphocyte analysis suggest that anti-

bodies against CD45 be added to all tubes as a third color to be used

for gating. By gating on CD45 positivity along with SSC, the gated

cells (Fig. 6.14) will not include high numbers of erythrocytes, debris,

or platelets (all CD45 negative). In this way gating can be re®ned if

you are using a ¯ow cytometer with extra ¯uorescence parameters.

The distinction between lymphocytes and monocytes is not, however,

helped by this procedure (and the CD45/SSC gate in Fig. 6.14 in-

dicates ambiguity here). Figure 6.15 illustrates CD45/SSC gating in a

bone marrow sample, where this type of analysis has proved partic-

ularly useful in identifying clusters that are related to various mature

and immature hematological lineages.

In fact, this CD45/SSC gating marks the general trend away from

using scatter parameters toward a quite di¨erent strategy for ¯ow

analysis. The basic problem, as seen above, is that lymphocytes (or

any other taxonomic group) are not a homogeneous collection of

cells with perfectly delineated physical characteristics; they are mainly

homogeneous, but they usually contain at least some cells at the

fringes with marginal characteristics. Any gating based on FSC/SSC

forces us either to exclude these fringe cells or to include many ex-

traneous cells. With the availability of multicolor instrumentation

Fig. 6.14. The use of CD45 to exclude erythrocytes, platelets, and debris from a

lymphocyte gate. The distinction between monocytes and lymphocytes can be am-

biguous with either the FSC/SSC or the CD45/SSC gate. Data ®le provided by Marc

Langweiler.

Leukocytes, Surface Proteins, and the Strategy of Gating 109

and multicolor stains, it has become possible to avoid making any of

these di½cult gating decisions and to include all cells in the analysis

by using stain itself either to gate in or to gate out the cells of interest.

For example, if one is studying the prevalence of a particular

subpopulation among lymphocytes, one could stain every peripheral

blood mononuclear cell sample with a PE stain for monocytes in

addition to an FITC conjugate of the marker of interest. Then, in the

analysis stage, one could simply gate out (exclude) from analysis any

PE-positive particles and analyze all the PE-negative particles for the

percentage that are FITC-positive. Figure 6.16 shows an example

of this protocol. By the use of three-color analysis, there are even

greater possibilities. This type of analysis avoids the necessity of prior

decisions about the FSC and SSC characteristics of the cells of choice

(e.g., lymphocytes). It becomes particularly important when the cells

of interest are less homogeneous in physical characteristics than lym-

phocytes. For example, by gating on a stain that is speci®c for cyto-

keratin (a protein found on tumor cells of epithelial origin), tumor

cells within a mixed population from a breast tumor biopsy specimen

can be selected for further analysis (e.g., DNA content) without any

prejudgement about the FSC or SSC of a poorly de®ned and hetero-

geneous population of abnormal cells.

Fig. 6.15. Bone marrow from a normal donor showing CD45/SSC clusters.

CD45 expression varies as cells mature. Lymph  lymphocytes; Mono  mono-

cytes; L-Blast  lymphoblasts; M-Blast  myeloblasts; Myeloid  neutrophils; and

Erythroid  cells of the erythroid lineage. From Loken and Wells (2000).

Flow Cytometry110

A logical extension of this kind of technique can be seen in the

methodology proposed by PK Horan in 1986: No decisions based on

the scatter characteristics of cells have been made. Cells are stained

simply with a cocktail of conjugated monoclonal antibodies at ap-

propriate (sometimes not saturating) concentrations, and all the cells

in the mixture are classi®ed according to their staining characteristics

(Fig. 6.17). Staining cells from leukemic patients currently follows a

similar strategyÐwhere abnormal and normal cells are de®ned by the

way they cluster in a two-dimensional dot plot. In other words, cells

are de®ned by their relative intensities more than by their negativity

or positivity for a given antibody.

At the beginning of this section, a strategy was described for plac-

ing a scatter (FSC/SSC) gate and then evaluating it in terms of purity

and inclusivity. We were then forced to admit that gating is often an

uneasy and subjective compromise between these con¯icting criteria.

We ®nd therefore that we must conclude that using FSC and SSC

characteristics to gate cells may not be a good thing after all. The

availability of multicolor analysis has led to a trend toward using

staining characteristics to de®ne the cells of interest without regard to

Fig. 6.16. Rather than a scatter gate, a PE-anti-CD14 stain can be used to exclude

monocytes from a count of B and non-B lymphocytes. Data courtesy of Jane

Calvert.

Leukocytes, Surface Proteins, and the Strategy of Gating 111

their possibly variable physical (light scatter) properties. By using one

or more colors in the analysis either to select or to exclude (gate in or

gate out) particular groups of cells, we can avoid any prejudgement

on the physical characteristics of those cells. This overall strategy

makes sense when our system allows for multicolor analysis (with

parameters to spare) and when antibodies are available to de®ne the

cells of interest. It is, nevertheless, still true that antibodies to de®ne

subsets of cells are not perfect: Cells of di¨erent phenotypes react

with similar antibodies (sometimes, thankfully, at di¨erent intensities),

and several antibodies are often required to fully de®ne the taxonomy

of a cell.

As a summary comment on gating, we need simply to remember

that in ¯ow cytometry our questions are usually formulated in terms

of ``what percentage of a certain population of cells is positive for a

certain set of characteristics?'' The choice of a gate de®nes that ``cer-

tain population'' of cells. The choice of that gate will therefore a¨ect

the answer to the question (the percentage positive). Whether gating

20 30

log green fluorescence intensity

log red fluorescence intensity

40 50

NK cells

cells

B cells

Fig. 6.17. The two-color ¯uorescence pro®le of peripheral blood mononuclear cells

stained simultaneously with six di¨erent monoclonal antibodies to delineate ®ve dif-

ferent populations of cells. From Horan et al. (1986).

Flow Cytometry112

is applied by means of scatter characteristics, by means of staining

characteristics, or not at all, the procedure still needs to be described

and quanti®ed if the results are to be meaningful and reproducible. It

is only when we have stated exactly which ``certain population of

cells'' we are analyzing that we have ful®lled our goals of objectivity

and/or explicit subjectivity in ¯ow analysis.

FURTHER READING

Chapters 3 and 5 in Ormerod, Chapters 3.2 and 3.3 in Diamond and

DeMaggio, Chapters 10 and 11 in Darzynkiewicz, Chapter 6.2 in Current

Protocols in Cytometry, and Chapters 17 and 34 in Melamed et al. are all

good discussions of general lymphocyte staining methodology for ¯ow

analysis.

Chapter 1.3 in Current Protocols in Cytometry and Chapter 14 in Darzyn-

kiewicz (both by Robert Ho¨man) are excellent discussions of sensitivity

and calibration.

Volume 33, number 2 (1998) of Cytometry is a special issue devoted to

``Quantitative Fluorescence Cytometry: An Emerging Consensus.''

Leukocytes, Surface Proteins, and the Strategy of Gating 113

Cells from Within:

Intracellular Proteins

Although many applications of ¯ow cytometry involve the staining of

cells for proteins expressed on the outer membrane, cells also have

many proteins that are not displayed on their surface. With appro-

priate procedures, ¯ow cytometry can provide a means to analyze

these intracellular proteins. The outer cell membrane is impermeable

to large molcules like antibodies; however, if we intentionally ®x cells

to stabilize proteins and then disrupt the outer membrane, the cells

can be stained with ¯uorochrome-conjugated monoclonal antibodies

against intracellular proteins. After time to allow the antibodies to

pass through the now-permeabilized membrane, the cells are washed

to remove loosely bound antibodies and then are run through the

¯ow cytometer to measure their ¯uorescence intensity.

This intensity should, under good conditions, be related to the

amount of the intracellular protein present. However, in describing

our ability to stain cells for surface proteins, we mentioned that it is

best to stain viable cells. Dead cells have leaky outer membranes;

they often show high nonspeci®c staining because antibodies get

through the disrupted membrane and become trapped in the intra-

cellular spaces. Therein lies a con¯ict in our ability to stain cells for

intracellular proteins. Because antibodies of all types are easily

trapped in the cytoplasm, there is greater potential for nonspeci®c

staining of permeabilized cells than intact cells. The very procedure

that we carry out to give access of the staining antibody to its target

(intracellular) antigen actually increases the access of all antibodies

to nonspeci®c targets. To lower this nonspeci®c background, antibody

titers are critical and washing steps are important. Unfortunately,

115

Flow Cytometry: First Principles, Second Edition. Alice Longobardi Givan

 2001 by Wiley-Liss, Inc.

ISBNs 0-471-38224-8 (Paper); 0-471-22394-8 (Electronic)

even with low antibody concentrations and careful washing, back-

ground ¯uorescence from isotype-control antibodies is often consid-

erably higher on permeabilized than on intact cells.

There is, in addition, a second problem. The procedures used for

®xing and permeabilizing cellsÐto give the staining antibodies access

to intracellular proteinsÐcan modify or solubilize some antigens,

thus destroying the stainability of the very proteins that are being

assayed. To make matters worse, the protocol that works best for one

antigen may entirely destroy a di¨erent antigen. This should not be

surprising after consideration that ``intracellular'' includes proteins of

many types and in many di¨erent environments. Some intracellular

proteins are soluble, some are bound to organelle membranes, and

some are in the nucleus. Therefore, methods for staining cells for

intracellular proteins cannot be as standard or as dependable as the

methods for staining surface proteins. They have to be individually

optimized for the cells and the proteins in question.

METHODS FOR PERMEABILIZING CELLS

While not attempting to describe possible methods in detail, I feel it is

important here to point out the issues involved in intracellular stain-

ing because they highlight some general issues that a¨ect all of ¯ow

cytometric analysis. Methods for permeabilizing and ®xing cells are

various and must be optimized for the particular intracellular antigens

being detected because some antigens are more robust than others in

the face of di¨erent agents. Figure 7.1 gives an example comparing

®xation/permeabilization e¨ects on two di¨erent intracellular anti-

gens: Five di¨erent ®xation/permeabilization protocols have been

used, and their e¨ects on staining PCNA (proliferating cell nuclear

antigen) and p105 (a mitosis-associated protein) have been compared.

The good news is that you can stain for intracellular antigens. The

bad news is that it may be di½cult to stain cells optimally for

two di¨erent antigens at the same time (and the relative intensity of

staining for two di¨erent antigens may tell you little about the actual

relative proportions of these proteins in the cell).

The general protocol for intracellular staining involves, ®rst,

staining the cells for any surface (outer membrane) antigens, as de-

scribed in the previous chapter. Then the surface proteins with their

Flow Cytometry116

bound antibodies, as well as the intracellular proteins, are ®xed gently

to stabilize them. The purpose of the ®xation is to cross-link the

proteins well enough that they are not removed or washed out of the

cells after the cells are permeabilized, but not so well that the intra-

cellular antibody binding sites are masked or destroyed. Although

ethanol and methanol can be used for ®xation (by themselves or

following another ®xative), the most common ®xative used prior to

intracellular staining is formaldehyde. Formaldehyde is generally

used at lower concentration and/or for a shorter period of time than

for routine ®xation of surface-stained cells (where ®xation overnight

in 1% formaldehyde is the [optional] last step of the procedure before

¯ow cytometric analysis). Formaldehyde (at 0.5±1.0%) for 10 min is

a good suggested concentration and time for cell ®xation, but lower

or higher concentrations, for shorter or longer periods of time, might

be required.

Fig. 7.1. The e¨ects of di¨erent ®xation protocols on the relative amounts detected

of two di¨erent intracellular proteins. Modi®ed from A McNally and KD Bauer as

published in Bauer and Jacobberger (1994).

Intracellular Proteins 117

This formaldehyde ®xation does permeabilize the cytoplasmic

membrane a bit (formaldehyde-®xed cells are permeable to small

molecules), but proteins are often cross-linked too tightly for staining

of intracellular proteins with antibodies. Therefore the ®xation step

is followed by a permeabilization step. Permeabilizing agents are

usually detergents, such as Triton X-100, digitonin, NP40, or saponin,

at concentrations of about 0.1%. Combined ®xation/permeabilization

reagents are also available as proprietary commercial reagents. With

luck, the detergent will open up the cell enough so that the now-®xed

proteins are accessible to the antibodies used for staining.

What are the criteria by which we can determine whether a ®xation/

permeabilization procedure has been optimized for an antigen in

question? This optimization is, in essence, no di¨erent from opti-

mization of a protocol for surface staining of cells. It is ®rst necessary

to maximize the ¯uorescence intensity of cells that are known to pos-

sess the intracellular antigen (the positive control); ®xation time and

concentration need to be altered in combination with di¨erent deter-

gent concentrations to increase the positive staining. It is then neces-

sary to decrease the background staining (using cells stained with

isotype-control antibodies) as much as possible; this is done by trying

increasing detergent concentrations and washing the cells thoroughly

in bu¨er that contains the detergent. In other words, the goal is to

increase the signal-to-noise ratio. Because antibody concentration,

®xative agent, ®xative concentration, ®xation time, choice of per-

meabilization agent, and concentration of that permeabilizing agent

are all variables in this protocol (and the optimal characteristics of

each may be di¨erent for di¨erent antigens), staining for intracellular

antigens requires some persistence on the part of the investigator. The

following examples (in this chapter and in the following chapter on

DNA) will demonstrate, however, that it is certainly possible.

EXAMPLES OF INTRACELLULAR STAINING

From the point of view of a ¯ow cytometer, surface, cytoplasmic, and

nuclear proteins are similar. The ¯ow cytometer cannot ascertain the

location of the source of ¯uorescence. In addition, the nuclear mem-

brane has large enough pores that it provides little or no obstacle to

staining once the outer, cytoplasmic membrane has been breached.

Flow Cytometry118

Cells have been stained successfully for nuclear proteins related to

proliferation (for example, PCNA, Ki-67, and various cyclins, which

will be discussed in the chapter on DNA) and to tumor suppression

(for example, p53, c-myc, and the retinoblastoma gene product).

They have also been stained for proteins bound to interior membrane

surfaces (e.g., Bcl-2, multidrug resistance protein [MDR], and P-gly-

coprotein), and many strictly cytosolic proteins have been analyzed

(like tubulin, hemoglobin, surface proteins that exist intracellularly at

various stages of di¨erentiation, and many cytokines).

As an example of one of the more complex biological situations,

we can use the staining of cytokines as an illustration. Cytokines are

a diverse class of proteins that, in response to cell stimulation, are

synthesized and then secreted by leukocytes. For example, when T

lymphocytes are stimulated, either nonspeci®cally or by immuno-

logical triggers, they begin to synthesize interferon-g in their endo-

plasmic reticulum, send the proteins to the Golgi apparatus, and then

secrete the molecules into the environment for stimulation of neigh-

boring cells. To stain for intracellular interferon-g, the usual technique

is to stimulate cells with a biological trigger and then to incubate them

with an inhibitor (brefeldin A or monensin) for several hours. These

inhibitors block the normal secretion of proteins from the Golgi

apparatus and thus allow the cytokine concentration to build up in

the cell to levels that are detectable. After the incubation period, the

cells are stained for any surface antigens of interest, ®xed brie¯y in

formaldehyde, permeabilized with saponin, and, ®nally, stained with

a monoclonal antibody against interferon-g.

Figure 7.2 shows an example of the way in which cells can be

stained for a phenotypic surface marker (CD8) as well as the intra-

cellular cytokine, interferon-g. The ¯ow data indicate that interferon-

g is associated, after PMA-ionomycin stimulation, primarily with

CD8-negative cells. More of the CD8-negative than the CD8-positive

cells have intracellular interferon-g, and those that have that cytokine

have more of it per cell. The tricks in the procedure for staining in-

tracellular cytokines are as much biological as chemical (because the

stain is for the end result of a functional process). In addition to a

knowledge of how to ®x and permeabilize a cell and how to avoid

nonspeci®c staining, we require knowledge of how to trigger the

cytokine production, knowledge of the time course of cytokine syn-

thesis after stimulation, and knowledge of how long cells can survive

Intracellular Proteins 119