9 Flow Cytometric Strategies to Study CNS Development

7/23/2019 9 Flow Cytometric Strategies to Study CNS Development

http://slidepdf.com/reader/full/9-flow-cytometric-strategies-to-study-cns-development 1/32

Flow Cytometric Strategies

to Study CNS Development

Dragan Marie, lrina Marie, and leffery 1. Barker

I, Introduction to Flow Cytometry

The technique of flow cytometry was initially developed to

count and size particles. However, it has progressively evolved

into a sophisticated analytic tool for rapidly quantifying multiple

properties of individual cells or cellular constituents in suspended

nonhomogeneous populations. All flow cytometry instruments

share a common feature: single cells or particles are pressured to

flow through a sensing region in which their electrical resistance

or optical properties are recorded. Most commonly, these proper-

ties are visualized with fluorescent molecules that bind specifi-

cally to the biological constituent(s) to be measured. Typically,

these fluorescent molecules are excited by laser beam(s) tuned at

specific wavelenghtts) and their emission(s) collected with an array

of appropriate filters that convey the signals to photomultiplier

tubes and ultimately to a computer.

Flow cytometry complements other optical and electrical

recording strategies that have recently evolved and offers clear

advantages, including the acquisition of multiple parameters at

very high rates (1000-3000 events/s), objectivity, and powerful

sorting capabilities. Over the last 25 yr, it has become widely used

in the fields of hematology, immunology, oncology. and microbi-

ology. Cell counting, identification and classification, cell cycle

studies, measurements of DNA content and cell proliferation, chro-

mosomal karyotyping, and studies of cellular physiology are

among the most widespread research and clinical applications of

flow cytometry (Melamed et al., 1990).

From

Neuromethods, vol 33 Cell Neurohology Techques

Eds A A Boulton, C B Baker, and A N. Bateson 0 Humana Press Inc

287



288 Marie, Marie, and Barker

In the field of developmental neurobiology, however, flow

cytometry has not been extensively used so far. In this chapter,

we demonstrate several possible applications of flow cytometry

in the studies of CNS development: rapid identification of spe-

cific cell populations in the developing CNS using multiple surface

and cytoplasmic markers putatively specific for neuroepithelial,

neuronal, and glial cell lineages; analysis of cells in specific stages

of cell cycle and apoptosis; physiological recordings of membrane

potential and cytosolic calcium and pharmacological discovery

of functional receptors and ion channels; and precise isolation and

sorting of distinct cell populations, based on a specific epitope

expression or a functional response.

2. Cell Preparation

One of the most crucial steps in using flow cytometry to inves-

tigate physiological and pharmacological properties of develop-

ing CNS at a single-cell level is cell preparation. Cells composing

the CNS during embryonic (E) and early postnatal (P) periods can

be most completely dissociated into single cell suspensions by

enzymatic digestion with papain (Huettner and Baughman, 1986,

Marie et al., 1997). Other commonly used dissociation protocols,

including mechanical (Mandler et al., 1988), trypsin (Schaffner and

Daniels, 1982), and collagenase (Johnson and Argiro, 1983) can

lead to highly variable cell recoveries, which are associated with

up to 50 reduction in cell yield, together with a markedly

decreased cell viability (Marie et al., 1997).

In our study, the papain dissociation protocol was as follows

Embryonic (Eli-22) and early postnatal (PO-7) CNS tissues were

quickly dissected into telencephalic (Eli-14) and neocortical

(E15-P7), olfactory bulb, hippocampal, thalamic, hypothalamic,

mesencephalic, rhombencephalic, and spinal cord regions (Hebel

and Stromberg, 1986; Altman and Bayer, 1995) and immediately

placed in ice-cold saline to retard further developmental changes.

Tissues were cleaned, minced with forceps, and then completely

dissociated into single-cell suspensions by the enzymatic action

of papain (20 U/mL) for 30-45 min at 37”C, and gentle trituration

as described (Huettner and Baughman, 1986). In some experi-

ments, 350~pm thick coronal sections of late embryonic neocortex

were first microdissected along the incipient white matter into

cortical plate/subplate (CP/SP) zone, including layer I cells, and



CNS Development Studies

289

ventricular/subventricular (VZ/SVZ) zone, including lower in-

termediate zone (IZ) cells, and then dissociated as described. This

protocol routinely yielded single-cell suspensions with greater

than 95 vitality as determined by trypan blue exclusion on the

microscope stage and confirmed using vital (acridine orange) and

nonvital (propidium iodide) dye staining of cell suspensions

analyzed by flow cytometry.

After isolation, the cells were labeled with fluorescent antibod-

ies and/or indicator dyes, and passed through the laser-based fluo-

rescence activated cell sorter (FACS), where up to five different

parameters of each single cell (including cell size and complexity,

and immunocytochemical, membrane potential and calcium fluo-

rescence signals) were measured simultaneously, at the rate of

several thousand cells per second. In some experiments, precise

sorting of different cell subpopulations then followed, based on

any one or a combination of these different cell parameters. A sche-

matic outline of the method and some of the cell properties that

can be quantified with different indicators by flow cytometry are

depicted in Fig. 1.

All recordings were carried out with a FACSTAR’ flow cytom-

eter (Becton Dickinson, Mountain View, CA). Cells were excited

using an argon ion laser (Spectra Physics, Model 2016, Mountain

View, CA) operated at 500 mW and tuned to 488 nm. Forward

angle light scatter (FALS), a property related to cell size, and dif-

ferent fluorescence emissions of individual elements were

randomly recorded at 1000-2000 events/s. This rate of data

acquisition allowed profiling the properties of approx 10,000 cells

in 5-10 s. FALS data were collected in a linear mode using a com-

bination of 488 + 10 nm bandpass and neutral density filters,

whereas fluorescence emissions were logarithmically amplified

and filtered at appropriate wavelengths. In multiple labeling

experiments, fluorescence emissions were corrected for color cross-

over by using electronic compensation. FALS properties and fluo-

rescence intensities were each resolved into 1024 channels. The data

were analyzed using Cell Quest Analysis software operating on a

FACStation Macintosh-based computer platform (Becton Dickinson).

3. lmmunocytochemistry

One of the major difficulties encountered when studying the

development of the CNS is the inability to readily identify specific



29



CM Development Stud/es 29’1

cell lineages at distinct phases of proliferation and differentiation.

One of the reasons IS the lack of availability of uniquely specific

cell markers. There is at present a rapidly growing number of com-

mercially available polyclonal and monoclonal antibodies that can

be used to detect specific cell surface, cytoplasmic, or nuclear

epitopes in CNS cells. However, many of these epitopes are shared

among neuroepithelial, neuronal, and glial cell types at some

stages of their development. Therefore, there is an increasing need

for using double- and triple-immunostaining procedures, in order

to obtain a more precise identification of specific cell populations

under mvestigation. A flow cytometer equipped with dual and

triple emission filter sets is ideally suited to access this complex-

ity and diversity of specific CNS populations in a very rapid and

precise manner. In the following sections, we describe the identi-

fication of putative neuroepithelial, nerve- and glia-specific mark-

ers on several populations of acutely dissociated embryonic and

early postnatal CNS cells using flow cytometry and double- or

triple-immunolabeling protocols with specific antibodies against

cytoplasmic and plasma membrane epitopes.

3.1. lmmunocharacterization by Cytoplasmic Markers

Different cytoplasmic markers tagged with fluorochrome-con-

jugated antibodies can be identified by flow cytometry, but prior

cell fixation and membrane permeabilization are necessary. Laser-

based flow cytometry is more sensitive in detection of immuno-

Fig. 1. ~~WZVOUSage)Accessing CNS development by flow cytometry

(A) In order to study the biological properties of developing neuroeplthe-

hal, neuronal,

and glial cell lineages during CNS development, the cells

first have to be dissociated into uniform single-cell suspensions. (B) The

cells are then immunoreacted, stained or loaded with reagents that target

their distinct phenotyprc or physiological properties The labeled cells are

passed through a nozzle tip (with an aperture of 70 pm> and Illuminated

one at a time with a laser set at a desired excitation wavelength. Then

light-scattering and fluorescence emission properties are collected with

an array of specific filters connected to their respective photomultiplier

tubes, which convey the signals to the computer. (C) By vibrating the

nozzle tip at high frequencies (typically 24,000 Hz) and electronically

charging the mdivrdual droplets of salme in which each cel l is suspended,

It is possible to sort specific populations of cells based on a distmct combr-

nation of then light scattering and fluorescence emission properties.



292 Mar/c, Marie, and Barker

fluorescence signals than conventional lamp-based fluorescence

microscopy and offers the advantage of precise and objective elec-

tronic quantification of different fluorescence intensities in tens

of thousands of cells virtually all at the same time.

This is particu-

larly important in studies on the developmental appearance and

disappearance of different cell markers, since it is often difficult

to distinguish precisely between background and very low

immunopositive signals with conventional methods. Figure 2 rep-

resents the results obtained after immunostaining acutely disso-

ciated and ethanol-fixed El9 neocortical cells with a rabbit

polyclonal class IgG anti-nestin antibody, an intermediate filament

protein associated with neuroepithelium-derived progenitor cells

(Hockfield and McKay, 1985) (a gift from R. McKay, NIH,

Bethesda, MD), and a mouse monoclonal class IgG anti-MAP2

antlbody, a neuronal cytoskeletal marker (Sigma, St. Louis, MO).

For flow cytometry, these immunoreactions were respectively

visualized with a phycoerythrin (PE)-conjugated goat anti-rabbit

IgG and biotinylated goat anti-mouse IgG (Jackson Immuno-

Research Laboratories Inc., West Grove, PA), followed by

Fig. 2. (opposzte page) Double immunolabeling of cytoskeletal markers

in fixed cell preparations. (A) Flow cytometric assessment of antmestm

and anti-MAP2 immunostaming of El9 neocortlcal cells reveals four

distinct subpopulatlons. Nestin- /MAP2-, Nestm+/MAP2-, Nestin-/

MAP2+, and Nestin+/MAl?? Whereas most of the Nestin+/MAP2- and

Nestm/MAP2+ cells are located m the VZ/SVZ and CP/SP, respectively,

both regions contain Nestin+/MAlY subpopulations. However, there

are marked region-specific fluorescence intensity differences in both

cytoskeletal markers between these two subpopulations Nestmh’gh/

MAP2’“” expressors are located in the VZ/SVZ and Nestin’ow/MAP2t”~h

expressors appear m the CP/SP (B) Immunostainmg of acutely plated

CP/SP and

VZ/SVZ cells with the same antibodies clearly reveals

MAl’2h’gh immunopositive cells in the CP/SP and Nestinh’gh cells m the

VZ/SVZ, whereas the quantification of Nestn+ and MAP2“‘” subpopu-

lations IS somewhat ambiguous using the light microscope (C) Immun-

ostaining of the El9 coronal sections of the cortex under identical

conditions used for flow cytometry confirms that nestin-lmmunoposltlve

cell bodies are for the most part located in the VZ/SVZ, whereas MAP2-

lmmunopositlve cells are present mainly m the CP/SP However, tissue

sections can not resolve the intensity differences of either cytoskeletal

marker m indiv idual cells.



Anti-Nestin-PE

B

C

Anti-Nestin

Anti-MAP2

h..

Anti-Nestin

I

CP

SP

IZ

svz

vz

Anti-MAP2

I.

I

i,, .

i

Fig. 2(A-C)

293



294 Max, Mar/c, and Barker

streptavidin-Red670 (Life Technologies Inc., Gaithersburg, MD).

Cell immunofluorescence characteristics were acquired using a

488 nm laser excitation and fluorescence filters set at 575 rt 25 and

670 _+ 20 nm to detect PE and Red670 emissions, respectively.

Reactions in acutely plated cells were visualized with appropri-

ate blotinylated secondary antibodies, followed by streptavidin-

peroxidase (Jackson ImmunoResearch Laboratories Inc., West

Grove, PA) and the development of peroxidase reaction product

in 3-amino-9-ethylcarbazole (AEC) containing 0 001 HzO,.

Quantitative flow cytometric assessment of logarithimlcally-

amplified anti-nestin and anti-MAP2 immunofluorescence inten-

sities revealed different levels of nestin and MAP2 expression in

neocortlcal cells, as some transformed from progenitor stages in

the VZ/SVZ to more differentiated neuronal stages in the CP/SP

(Fig 2A). We akl y expressing nestin- and MAP2-immunoposltive

cells comprised distinct subpopulations in the flow cytometric

recordings, although they could not be easily accounted for under

the microscope (Fig. 2B), despite the fact that the percent of high-

expressing immunopositive cells obtained with both methods was

quite similar. For example, it was very difficult to precisely quan-

tify the large population (approx 50 ) of nesti@” positive cells in

the CP/SP dissociates without a flow cytometer, even when the

antibody reaction in acutely plated cells was visualized with a

much more sensitive enzymatic endpoint, instead of a fluorescent

endpoint. Because of this objective, extremely sensitive and rapid

data acquisition, the results obtained with flow cytometry are often

more complete compared to the results obtained with conventional

microscopy techniques.

3.2. lmmunocharacterization by Cell Surface Markers

Living CNS cells in different stages of neuronal and glial lin-

eage progression can be identified using antibodies against dis-

tinct cell-surface markers. A variety of monoclonal antibodies are

now available that recognize specific ganghosides and other

epitopes on the plasma membranes of developing CNS cells. In

our studies, we have used a mixture of tetanus toxin fragment C

(TnTx) and a mouse monoclonal class IgG anti-TnTx antibody, a

marker of terminally postmitotic developing neurons (Koulakoff

et al., 19831, a mouse monoclonal class IgM anti-A2B5 antibody, a

neuronal and O-2A progenitor marker (Abney et al., 1983), a mouse

monoclonal class IgM anti-04, and a mouse monoclonal class IgG




295

anti-galacto-cerebroside (GalC) antibodies (Boehringer Mannheim

Biochemicals, Indianapolis, IN), two markers of early and late

stages of oligodendrocyte lineage development (Raff et al., 1978,

Schachner et al., 1981). Acutely dissociated cells were double

labeled with different combinations of these antibodies and pri-

mary immunoreactions were then visualized by immunostaininmg

with PE-conjugated goat anti-mouse IgM antibody and a bio-

tinylated goat anti-mouse IgG (Fey fragment specific) antibody

(Jackson ImmunoResearch Laboratories, West Grove, PA) fol-

lowed by streptavidin-Red670 (Life Technologies, Gaithersburg,

MD). Results of TnTx/A2B5 and GalC/04 double-immuno-

staining reactions at several ages and regions during CNS devel-

opment revealing qualitative and quantitative differences in

expressions and coexpressions of these surface epitopes are pre-

sented in Fig 3.

4. Assay of Proliferative and Apoptotic Potentials

of Neocortical Subpopulations

It is well accepted that development of the CNS system involves

both cell proliferation and naturally occurring cell death, or

apoptosis (Naruse and Keino, 1995). Here we show that these pro-

cesses can be expeditiously detected and quantified by flow

cytometry using fluorescently labeled antibodies against thymi-

dine analog bromodeoxyuridine (BrdU), a marker of S-phase cells

(Gratzner, 19821, annexin V, an anticoagulant protein that prefer-

entially binds to phosphatidyl serine phospholipids exposed on

the outer leaflet of the cytoplasmic membrane early in apoptosis

(Koopman et al., 1994; Martin et al., 1995), and propidium iodide

(PI), a fluorescent dye that binds to all double-stranded nucleic

acids and can be used to measure total DNA content (Dolbeare

et al., 1983).

4.1. Detection of BrdU Incorporation

by DNA -replicating Cells

Timed pregnant dams at embryonic day 16 were given a single

intraperitoneal injection of BrdU (50 ug/g body weight) (Sigma)

and sacrificed 60 min later. The pups were removed and several

regions of the developing CNS acutely dissociated as previously

described. Detection of BrdU incorporation was conducted by

permeabilizing the ethanol-fixed cells with 2 N HC1/0.5 Triton



296

Marie, Marie, and Barker

Neocortex K P/SP) Neocortex NZISVZI

32 3

I 1

Spinal Cords

PI

Anti-A2BS-PE Fluorescence

Neocortex Olfactory Bulb

Hippocampus

Rhombencephalon

1 7

t I

I7

Cerebellum

hl

1

Anti-040PE Fluorescence

Fig. 3(A,B)




297

X-100 and immunoreacting the exposed DNA with FITC-conjugated

mouse anti-BrdU monoclonal antibody (Becton Dickinson). Finally,

the immunoreacted nuclei were counter-stained for total DNA con-

tent by resuspending the cells in PBS containing 5 ug/mL PI.

Bivariate distributions of BrdU incorporation and total DNA con-

tent were then assessed on a single-cell level by flow cytometry

(Fig. 4). Upon excitation at 488 nm, the green (FITC-conjugated

anti-BrdU) and red (PI) fluorescence intensities emitted by each

cell were acquired using bandpass filters set at 530 + 30 and

575 + 20 nm, respectively. Electronic gating was used to exclude

any residual cellular aggregates, which consistently accounted

for ~5 of the total number of events. The percentages of BrdU+

(S-phase cells) and BrdU- subpopulations with diploid or tetrap-

loid DNA content (reflecting cells in GJG, and GJM stages of

the cell cycle, respectively) were quantified using a Cell Quest

data analysis system.

4.2. Detection of Plasma Membrane

and Nuclear Markers of Apoptotic Cells

Apoptotic cells can be first detected at the level of plasma mem-

brane using annexin V (Koopman et al., 1994; Martin et al., 1995).

Fig. 3 fprevzous page) Double immunolabeling of surface markers on

viable cell preparations quantified by flow cytometry. (A) Anti-A2B5

and anti-TnTx immunostaining of El9 neocortical cells reveals four dis-

tinct subpopulations. A2B5-/TnTx-, A2B5+/TnTx; A2B5-/TnTx+, and

A2B5+/TnTx+. Whereas all four subpopulations can be found in the pro-

liferative and early differentiating VZ/SVZ regions of the El9 neocor-

tex, the differentiating CP/SP region is for the most part composed of

A2B5-/TnTx+ cells, which we independently identified as a vrrtually pure

neuronal population using cytoskeletal markers and expressed morpho-

logical characterrstics m short-term cultures (see Fig. 2). Other mvesti-

gated CNS regions at El9 reveal a variable presence of all of the above

populatrons with the exception of the hippocampus, which notably lacks

A2B5-immunopositive cells. (8) Anti-04 and anti-GalC immuno-

reactions of P6 neocortical cells also reveal 4 distinct subpopulations.

04-/GalC-, 04+/GalC-, 04-/GalC+, and 04*/GalC+. Whereas 04+/GalC-

subpopulation 1s detected in all CNS regions tested at P6, the rhomben-

cephalic and spinal cord regions exhibit the greatest abundance of

04+/GalC+ cells, with the former also showing the greatest percent-

age of 04-/GalC+ cells.



298


HYPOTHALAMUS

CORTEX

Km $4

II n-. _<nLlm

e

RHOMBENCEPHALON

W-0

GdGv91.8

SPINAL CORD

lumbosacral (SC 1)

bl: 9.4

ag

SPINAL CORD

;

thoracic (SC t )

43

\ +*,

Gn/G1:86.0

Fig. 4. Three-dimensronal histograms of bivariate DNA data acquired

by flow cytometry illustrate an anatomical gradient of cellular prohf-

eratron and differentiation throughout the neuroaxis at Elk Relatively

few cells are actively syntheslzmg new DNA in the cervical spinal cord

and rhombencephalon, whereas cerebral cortrcal and lumbosacral spi-

nal cord populations exhibit the greatest percentages of cells m S-phase




299

In our studies we have triple-stained acutely dissociated El9 neo-

cortical cells with anti-A2B5-PE, anti-TnTx-Red670, and annexin

V-FITC and analyzed them by flow cytometry (Figs. 5A and B).

Green (FITC), orange (PE), and red (Red6701 fluorescence emis-

sions were simultaneously measured with bandpass filters at

530 _+30,575 A 20, and 670 + 20 nm, respectively. Percentages of

single-, double- and triple-positive and negative cells were quanti-

fied with the Cell Quest Analysis software using a logical gating

strategy.

Late stages of apoptosis were assessed by measuring total DNA

content of fixed cells stained with PI (Fig. 50, Cells double-

immunoreacted with A2B5-PE and TnTx-Red670 were sorted

based on their surface epitopes into A2B5-/TnTx-, A285+/TnTx-,

A2B5-/TnTx+, and A2B5+/TnTx+ subpopulations (seeSection 7.1.)

and fixed in 70 ethanol. The A2B5 and TnTx immunoreactions

were then stripped off the cell membranes in Triton/HCl solu-

tion and the cells further processed for detection of their total DNA

content (seeSection 4.1.). Late apoptotic or “A,,” cells were identi-

fied as cells with hypodiploid DNA content with respect to that

of G,/G, cells, which have diploid DNA. This cytometric prop-

erty, also referred to as “sub-G,/G, peaks,” depicting cells under-

going DNA fragmentation, has been shown to be a reliable marker

of cell death by apoptosis (Telford et al., 1991; Darzynkiewicz et

al., 1992; McCloskey et al., 1994).

5. Potentiometric Signals

of Dissociated Embryonic Neocortical Cells

One of the most crucial processes in CNS cytogenesis is the

development of membrane excitability. Although classical elec-

trophysiological techniques have been extensively used to char-

acterize membrane receptor/channel and ion properties of cells,

technical difficulties can be encountered in recording small, pro-

liferating, and immature cells, which constitute the majority of

cytoarchitecture during the earliest stages of CNS development

(reviewed by Barry and Lynch, 1991). In addition, microelectrode

techniques can be invasive to the cell membrane and only a lim-

ited number of cells can be recorded at any one time under the

same experimental conditions. One way of overcoming these dif-

ficulties is to use noninvasive techniques with fluorescent volt-

age-sensitive indicator dyes. Several studies have already reported



300


A2BS iTnTx

A2B5PE Immunofluorescence

Annexin V-FITC Fluorescence

A2BS fhTf

Propidium Iodide Fluorescence

Fig. 5. Flow cytometric assessment of early and late apoptotic neo-

cortical cells at E19: Al Live, unfixed cells were triple stained with anti-

A2B5 and anti-TnTx antibodies and annexin V-FITC. We have used

electronic gates depicted by the cross-hairs) on A2B5 and TnTx double-

immunoreacted cells to reveal their expression of annexin V. B) Annexin

V-FITC binding to the plasma membranes reveals a differential pres-

ence of annexin V’” and annexin Vhigh-positive cells in the four immuno-



CNS Development Stud/es

301

the utility of voltage-sensitive dyes in investigations of potentio-

metric signals of developing CNS tissues using digital video

microscopy (Walton et al., 1993) and flow cytometry (Mandler et

al., 1988; Krieger et al., 1991; di Porzio et al., 1993; Fiszman et al.,

1993). The advantage of these methods is that they allow experi-

mental access to the physiological properties of entire populations

of intact cells regardless of their size or morphology, and can pro-

vide a statistically complete account of potentiometric signals ran-

domly acquired from hundreds to thousands of individual cells

within a matter of seconds.

In our studies, we have used flow cytometry and oxonol, an

anionic voltage-sensitive indicator dye that partitions into the cells

according to membrane potential (Petit et al., 1993), to investigate

the development of membrane excitability throughout the embry-

onic CNS. After papain dissociation, cells were resuspended in a

physiological saline (in n-&I): 145 NaCl, 5 KCl, 1.8 CaCl,, 0.8 MgCl,,

10 HEPES, 10 glucose, and 1 mg/mL fatty-acid-free bovine serum

albumin (Sigma), the pH and osmolarity of which was adjusted

to 7.3 and 290 mOsm, respectively. Cells were then stained with

bis(l,3-dibutyl barbituric acid) trimethine oxonol (Molecular

Probes, Eugene, OR), a potentiometric dye that is negatively

charged at physiological pH. Since virtually all living cells exhibit

negative potentials, the dye’s negative charge opposes its cellular

accumulation at resting potentials, whereas depolarized cells stain

lo-fold or more relative to cells at rest. All of the potentiometric

results were recorded using 200 nM oxonol, since this concentra-

tion effectively stains cells well above autofluorescence levels.

Staining with oxonol requires approx 2 min to equilibrate at room

temperature, after which the mode and distribution of fluores-

cence signals remain stable for at least the duration of the typical

(Frg. 5, contznuedfvom prevtous page) identified subpopulatlons Separate

experiments using unfixed cells stained with annexm V-FITC and PI

revealed that only a few of annexin V’“” and the majority of annexin

Vh’~hcells were PI positive, demonstrating that membrane permeability

of most annexm Vi”” cells is not significantly compromised, thus mdi-

catmg the early phase

of

apoptosis. (Cl PI staming of sorted and fixed

A2B5-ITnTx-, A2B5+/TnTx-, A2B5-/TnTx+, and A2B5+/TnTx+ subpopu-

lations reveals a percentage of hypodiploid cells that positively corre-

lates with the percentage of annexin V-positive cells m the same

subpopulations




recording period (5-15 min). Oxonol-stained cells were passed

through a flow cytometer at a rate of 2000 cells/s, excited one at a

time by 488 nm and the resulting emissions detected with a single

filter set at 530 + 30 nm. A typical recording involved the acquisi-

tion of oxonol fluorescence intensities of 10,000 randomly sampled

cells recorded over 5 s, the distribution of which was then plotted

as a single-parameter frequency histogram (Fig 6).

5.1. Calibration of Membrane Potential

The relationship between oxonol fluorescence intensity and cell

membrane potential can be calibrated by recording the oxonol fluo-

rescence intensity profile under resting conditions and again after

exposing cells in the same test tube to gramicidin, a monovalent

cationophore previously used to relate oxonol fluorescence to theo-

retical membrane potential in spectrophotometric (Breuer et al.,

1988; MacDougall et al., 1988; Cruciani et al., 1991; Brent et al.,

1993) and flow cytometric studies (di Porzio et al., 1993). In elec-

trophysiological recordings, 1 PM gramicidin depolarizes cells to

0 mV in physiological saline. In our recordings, we have empui-

tally determined that saturating concentrations (21 PM) of grami-

cidin increase the fluorescence modes of oxonol-stained cortical

cells in a [Na+lO-dependent manner (Fig. 6). These results allowed

us to use a Goldman-Hodgkin-Katz formulation to relate oxonol

fluorescence to membrane potential and to calibrate the signals

Assuming that after gramicidin permeabilization total intracellu-

lar concentration of permeant Na’ and K’ cations remains con-

stant at approx 150 mM during the 10-s recording period, then

oxonol fluorescence modes of the signals in altered Na+O salines

can be related to membrane potential using a simplified Goldman-

Hodgkin-Katz equation in which the membrane potential is E, -

E0 = RT/ZF log [Na’ + K+ll/[Na+ + K+lO,where R, T, Z, and F have

their usual meanings. Modes of oxonol fluorescence can be cali-

brated in terms of membrane potential over much of the physi-

ological range, i.e., -90 mV-0 mV (Fig. 6B).

5.2. Survey of Membrane Excitability

in Developing Neurons

We have investigated the chemosensitivity of acutely dissoci-

ated El9 CP/SP neurons to saturating concentrations of various

neuroactive agents including acetylcholine, y-aminobutyric acid




303

Oxonohained

pmicidin-treated

’ [Na+l,bM) ’

0

145

V,,,= 0307FL,,-228

145

R = 0.99

7257

+I0

0

g -10

'i;j -20

P

$ -30

2 -40

aaf -50

P

0

-60

3 -70

-80

207 /

a

69

/

RMP

0

-loll~-~

400 450 500 550 600

650 700 750

4

800

Oxonol Fluoresce nce Intensity (Channels) Oxonol Fluoresce nce Intensity (Channels)

Fig 6. Calibration of oxonol fluorescence signals in terms of estrmated

membrane potential values. (A) Oxonol-stained neurons isolated from

the El9 CP/SP were resuspended in salines containing varying iNa+&

(145, 72.5, 20.7,6.9, and 0 n-M), which was replaced by equimolar con-

centrations of membrane impermeable N-methyl-D-glucamine. The cells

were then treated with 1 uM gramicidin and their fluorescence levels

recorded (B) The modal fluorescence values of the oxonol fluorescence

distributions (FL,,) m different [Na+10 are plotted against theoretical

membrane potentials (V,,,) as calculated by a simplified Goldman-

Hodgkin-Katz equation, The slope of this relationship reveals a rela-

tively constant conversion factor, which defined that a change in approx

3 fluorescence channel units in oxonol intensity is equivalent to 1 mV

change in membrane potential By substituting the modal oxonol fluo-

rescence value of El9 CP/SP neurons under our control resting condi-

tions for FL,,, we estimated the modal resting membrane potential of

these cells at -85 mV.

(GABA), glycine, kainic acid (agonist of a subtype of glutamate

receptors), and veratridine (agonist of voltage-dependent Na+

channels). Potentiometric responses were quantified in terms of

percentage of responsive cells and the amplitude of the responses,

which were, after calibration, converted into mV. After recording

a control profile, cells were exposed to different ligands and change

in oxonol fluorescence recorded after 2 min. All recordings were

performed at room temperature. The heterogeneity of excitatory

responses obtained in recordings of these cells (Fig. 7) indicate



304

Mar/c, Mar/c, and Barker

f; 1

: 9

B 60 : ’

z

5 40

c2

20

0

loo

1

.

0

:*

?,

0 *

I .J

::

0

: :

‘.

20

Membrane Potential (mV)

Fig. 7. Excitatory membrane potential responses of El9 CP/SP

neurons. Approx 90-95 of cells depolarize to either 10 PM GABA

or 100 pM veratridine, whereas less than 5 depolarize to 10 FM ace-

tylcholme. Veratrldme depolarizes cells to +20 mV, whereas GABA



CM Development Studies

30.5

that FACS potentrometry can serve as a powerful tool for the

investigation of the cellular distribution of functional receptor/ion

channels in different subpopulations of developing CNS tissues.

6. Intracellular Calcium Signals

of Dissociated Embryonic Neocortical Cells

Measurement of cytoplasmic calcium ([Ca”],) concentrations

in living cells is of great interest to many investigators, since Ca*+

is a ubiquitous second messenger throughout the CNS. Cazcc sig-

nals have been implicated in the development of many neuronal

and glial cell functions. Cell survival (Spitzer, 1994) and death

(Franklin and Johnson, 1994), neurotransmitter release (Hille,

1992), growth cone motility and neurite elongation (Mattson and

Kater, 1987), cell migration (Komuro and Rakic, 1992), synaptic

plasticity (Bear and Malenka, 1994), and regulation of gene

expression (Gallin and Greenberg, 1995) are only some of the Ca2+-

related processes that have been described. Investigation of the

mechanisms that regulate intracellular Ca2+ during histogenesis

of the CNS is therefore of crucial importance in understanding

the physiology of cell proliferation, migration, differentiation,

death, and cell-to-cell communication.

Initially, electrophysiological methods were used to study Ca2+

homeostasis and plasma membrane expression of Ca2+-selective

channels. However, recent development of a growing family of Ca*+-

sensitive fluorescence indicator probes has led to alternative opti-

cal and confocal microscopic strategies of recording Ca*+signals at

cellular and subcellular levels. One such probe is l-[2-amino-5-(2,7-

dichlor-6-hydroxy-3-oxy-9-xanthenyl) phenoxyl-2-(2’-amino-5’-

methyl phenoxy) ethanel-N,N,N’,N’-tetra-acetic acid or Fluo-3.

Flu03 is a fluorescein-derived Ca*+-sensitive dye that produces a

40-fold increase in fluorescence intensity upon bmding with free

(Fzg 7, confinuedfvom previous page) depolarizes virtually all cells to -40

mV. Only 50 of cells depolarize to -40 mV after exposure to 100 PM

glycme, whereas 100 PM kainic acid depolarizes approx 70 of cells

mainly to approx 0 mV. Gramicidin, which chemically clamps all cells

at 0 mV by permeabilrzing their plasma membranes with monovalent

cation-selective channels, is always used as a control at the end of each

experiment to reveal the fluorescence mtensrty and distribution of

potentrometrlc srgnals corresponding to 0 mV




cytosolic calcium (Minta et al., 1989). Although other types of Ca*+-

sensitive dyes have been described, e.g., quin-2, fura- and indo-l

(reviewed by Tsien, 1980; Grynkiewicz et al., 19851, the major advan-

tage of Fluo-3 lies in its high signal-to-noise resolution, low cytotoxic

and mitogenic properties as well as in optimal excitation properties,

at wavelengths in the visible as opposed to UV range (Tsien, 1989).

In our studies, we have used Flu03 in conjunction with flow

cytometry in order to reveal and characterize, on qualitative and

quantitative levels, the presence of functional intracellular Ca*+stores,

Na+-Ca*+ exchange mechanisms, and several voltage- and ligand-

stimulated Ca2’ channels in acutely isolated El9 CP/SP neurons. The

cells were loaded with 1 PM Flu03 for 45 mm at room temperature,

then washed and resuspended in physiological saline, passed through

a flow cytometer, excited one at a time by 488 nm and the resulting

emissions detected with a single filter set at 530 + 30 nm.

6.1. Calibration of [C’a2+lc

The fluorescence of Fluo-3-loaded cells is measured in arbitrary

intensity units, i.e., fluorescence channels, which can be converted

into estimated [Ca”], values after the calibration procedure is per-

formed (Kao et al., 1989) at the end of each experiment (Fig. 8). Fluo-

3-loaded cells were acutely treated with lo-20 PM ionomycin, a Ca*+

ionophore, and the resulting saturated levels of Flu03 fluorescence

then maximally quenched by the addition of 2 mM MnCl,. [Ca2+lcev-

els under resting and experimental conditions were calculated

according to the following equation: [Ca*+], = Kd x [F - F,,,] / [FmaX F].

Kd is defined as the dissociation constant for Ca2+-bound Flu03 and

is 400 nM at room temperature (Minta et al., 1989).

Fmax

represents

the maximum Flu03 fluorescence value, whereas F represents the

fluorescence value of cells under resting or experimental conditions.

FmIn s defined as the minimum Flu03 fluorescence in the presence

of saturating concentrations of MnCl,. Since Mn*+ ions readily dis-

place Ca*+ ons from Flu03 (Hesketh et al., 1983) and the Mn*+/Fluo-

3 complex is only one fifth as fluorescent as Ca*+ /Flu03 complex

(Kao et al., 1989; Minta et al., 19891,F,,, is then calculated as follows

(Vandenberghe and Ceuppens, 1990): F,,, = F,,, - (FmuX F,,,,,,) x

1.25. In our flow cytometric experiments, the fluorescence values of

F, Lx~ and h4na2

were defined as the arbitrary channel number of

the mode of Flu03 fluorescence distributions recorded from 10,000

randomly sampled cells under appropriate resting and experimen-

tal conditions (Fig. 8).




9000

8000

7000

8 6000

-"t 5000

cl

4000

3000

2000

1000

Flue-3luorescence Intensity Khannels)

F,,= 814

F

bfna2= 451

Fmln= 360

ICa2+lc = 400

0

400 440

l"" I "T'~l~~~~l-

400 520 560 600

640 600 720 760

Flue-3 Fluorescence Intensity (Channels)

rl

1100

Fig. 8 Calibration of Fluo-3 signals. Flu03 loaded El9 CP/SP neu-

rons were treated wrth 10 pM ionomycin for 2 min followed by 2 mM

MnCl, for an additional 1 min. F,,, and FMvlnCIzere measured from the

modal values of the resulting Fluo-3 fluorescence distributions. By sub-

stituting the modal Fluo-3 fluorescence value of El9 CP/SP neurons

under our control resting conditions for F, we estimated the modal rest-

ing lCaz+lc levels of these cells at approx 140 nM

6.2. Survey of the Contributors to Calcium Homeostasis

Regulation of Ca 2+ levels in most cells is achieved through

interactions of Ca2+ &ansport mechanisms in the plasma and

endo(sarco)plasmic reticulum membranes and Ca*+ buffering mecha-

nisms in the cytoplasm (Kostyuk and Verkhratsky, 1994). Ca2+ trans-

port in transmembranes is regulated by different Ca*+ channels, Ca*+

pumps, and Ca*+ exchangers, whereas intracellular Ca2+ stores and

Ca*+-binding molecules serve as a Ca2+-buffering system.

In our studies, we have surveyed the developmental expres-

sion of several mechanisms involved in Ca2+ homeostasis of El9

CP/SP neurons. intracellular calcium stores, Na+-Ca*+-exchange,

and voltage-sensitive calcium channels (VSCC) (Fig 9). Intracel-



308

Marie Max and Barker



V

ta

S

t

v

e

C

c

u

m

C

s

l

o

o

I

c

o

n

t

o

l

+

.

1

I

.

=

I

1

d

I

s

a

C

o

o

c

C

c

u

m

C

e

r

a

o

(

C

M

F

g

9

S

v

e

o

s

e

e

a

m

h

u

m

i

n

o

v

e

i

n

C

h

m

a

s

i

n

E

9

C

S

n

o

(

A

A

c

e

s

e

h

b

t

m

c

o

m

a

l

e

e

s

o

c

a

c

u

m

m

z

a

o

a

e

t

r

e

m

w

t

h

1

u

A

o

m

n

a

C

o

e

W

h

e

t

h

g

g

n

e

e

a

e

[

C

c

o

s

u

m

c

o

m

a

l

e

e

s

m

vr

u

y

e

e

y

c

e

r

e

o

d

o

y

6

a

3

o

c

e

s

r

e

p

t

o

c

a

e

n

a

r

y

a

n

s

m

a

o

r

e

p

v

e

y

(

B

R

u

p

o

i

n

N

O

r

e

s

a

n

p

o

e

a

o

1

n

M

n

e

e

o

C

c

n

h

m

o

t

y

o

c

e

s

a

a

m

c

o

m

a

i

n

e

e

o

C

n

h

r

e

m

n

n

5

o

h

c

e

s

S

e

e

p

u

e

o

h

sa

m

c

e

s

o

1

m

c

a

e

n

p

o

e

n

o

m

a

l

e

e

s

o

C

i

n

m

c

e

s

w

c

h

a

e

su

a

m

e

e

a

e

5

m

n

o

c

o

n

r

e

o

d

n

(

g

-

m

p

)

T

s

r

e

p

e

i

s

n

s

h

p

c

o

r

a

t

o

h

r

e

p

e

o

h

c

e

s

s

u

p

i

n

r

e

a

p

o

o

c

a

s

a

n

(

1

n

&

N

)

w

e

c

a

e

n

p

o

e

a

t

r

a

e

in

e

e

i

n

C

+

c

h

r

e

o

e

s

t

o

r

e

n

l

e

e

s

w

t

h

m

5

m

n

o

s

m

a

o

i

n

m

c

e

s

T

e

r

e

u

t

s

s

u

t

h

N

C

+

e

h

m

p

a

a

m

a

r

o

e

i

n

r

e

a

r

o

o

r

e

n

l

e

e

s

o

C

E

(

C

S

m

a

t

o

o

E

9

C

S

n

o

w

t

h

4

m

K

O

p

o

e

a

s

g

f

c

a

c

a

c

u

m

e

r

y

i

n

6

o

h

p

a

o

w

c

h

i

s

t

o

a

y

a

s

h

b

r

e

n

t

h

e

p

m

i

n

C

r

e

s

a

n

A

c

o

m

e

e

b

o

o

c

a

c

u

m

e

r

y

i

n

e

w

t

h

4

m

K

i

s

a

s

o

a

h

e

e

b

p

e

e

p

n

t

h

c

e

s

t

o

1

u

M

n

t

r

e

p

n

i

m

yn

t

h

v

r

u

y

a

ce

s

h

e

L

y

p

V

C

P

e

m

a

o

o

c

e

s

w

t

h

1

n

M

o

c

o

o

n

G

A

o

1

n

M

o

a

o

n

V

A

r

e

e

s

t

h

1

o

t

h

c

e

s

a

N

y

p

a

2

a

P

y

p

V

C

r

e

p

v

e

y




lular calcium stores were studied by resuspending the cells in Ca2+-

free saline and then stimulating them separately with 10 mM caf-

feine, 10 pM ryanodine, 10 pM thapsigargin, or 10 uM ionomycin

(Fig. 9A). Na+-C a*+-exchange mechanisms revealed under resting

conditions and after exposure to caffeine were studied by resus-

pending the cells u-t Na+O- ree salines (Fig. 9B). Functional L-type,

N-type, and P-type VSCC were individually revealed by expos-

ing the cells suspended in physiological salme to 40 mM K*O in

the presence of nitrendipine, o-conotoxin GVIA, or cu-agatoxin

VIA, respectively (Fig. 90. Due to the transient nature (lo-300 s

range) of the Ca2+c esponses obtained with some of the above con-

ditions, these recordings involved the acquisition of Ca2+csignals

at higher rates (3000 cells/s) than used in other experiments. In

addition, the “dead time” between application of the stimulus and

the recording was reduced to approx 2 s by using a Time Zero

module equipped with an injector system (Cytek Development,

Fremont, CA).

6.3. Caztc responses to neurotransmitter ligands

We tested Ca2+c esponses in El9 CP/SP neurons to asymptotic

concentrations of several neuroactive agents, including acetylcho-

line, GABA, glycine, kainic acid, and veratridine. The responses

were recorded using a Time Zero module as described above. After

recording a control profile, cells were exposed to different ligands

and changes in Fluo-3 fluorescence recorded after approx 2 s. Typi-

cal peak Ca2+c esponses, recorded at room temperature, are illus-

trated in Fig. 10.

7. Flow Cytometric Sorting

of Embryonic Neocortical Subpopulations

The studies of specific populations of the CNS in vitro are com-

plicated by our limited abilities to unequivocally identify and

expeditiously isolate pure cell types. Investigators commonly use

Fig. 10. (upposzte page) Survey of Ca2+c responses of El9 CP/SP neu-

rons to several neuroactrve ligands After the addrtion of 10

pM

acetyl-

choline, approx 70 of the cells exhibit an immediate submicromolar

rise in [Ca2+Jc that recovers to resting levels within 2 min of stimulation

(kinetics data not shown). At peak response, GABA and kainic acid m-

duce a Ca2+c rise in approx 60 of neurons to submrcromolar and micro-




Rcs(fllg

,f J

SO-

SO-

’ (

40-

0

100

OS ‘ 3 5 1 ZSIO

.

60-

I

; I

’ ,

40-

: :

00512

Cytosolic Calcium Concentration (PM)

molar levels, respectively, whereas glycine affects 30 of the cells,

elevating their Ca

2+c y 400 nM. Veratridine affects approx 90 of cells,

increasing [Ca’+] levels above 1 PM. Ionomycin typically mduces a maxI-

mum rise in [Ca5+lc in all cells recorded.



312 Max, Marie, and Barker

selective culture conditions to isolate neurons, astrocytes, oligo-

dendrocytes, and other cell populations. However, these meth-

ods usually require several days to weeks of culturing, during which

time cell properties may change and no longer reflect those

expressed in vivo. A variety of methods now exist that permit

enrichment of specific subpopulations based on surface epitopes (i.e.,

panning and complement lysis). However, these are complicated by

the fact that many antigenic epitopes are shared among different cell

types during development and hence a combination of markers is

required for the identification and isolation of specific cell subpopu-

lations. Using a flow cytometer equipped for sorting, it is possible to

isolate very pure specific cell subpopulations based on the presence

of multiple phenotypic or functional cell markers.

7.1. Sorting Based on Surface Epitope Expression

El9 neocortical cells were double immunostained with anti-A2B5

and TnTx antibodies, as described previously (seeSection 3.2.) and

categorized into four populations (TnTx+/A2B5-, TnTx+/A2B5+,

TnTx-/A2B5+, and TnTx-/A2B5-) based on their fluorescence sig-

natures determined by FACS electronic gates (Fig. 11, left most

panel). The four populations were sorted by means of electrically

charged saline droplets, which were deflected by charged plates

directly into appropriate test tubes (see Fig. 1). Sorted cells were

then washed twice in physiological saline and re-analyzed to test

for sorting purity, which was greater than 96 in all cases (Fig.

11, four right panels). After sorting, the viability of the cells

remained unchanged, with less than 5 trypan blue or PI-posi-

tive (dead or dying) cells in every sorted subpopulation.

7.2. Sorting Based on functional Response

Flow cytometers equipped for sorting also have a unique capa-

bility of isolating purified responding and nonresponding cell

populations based on sustained or transient functional responses

in different cells. El9 CP/SP neurons stained with oxonol or loaded

with Fluo-3 were stimulated with 100 pM kainic acid or 10 PM ace-

tylcholine, respectively. Responding and nonresponding subpopu-

lations were sorted based on a sustained membrane depolarization

induced by kainic acid or a transient calcium increase induced by

acetylcholine using electronic gates as shown in Fig. 12 (shaded

areas>. To test for the purity of kainic acid responding and




I I

I2

Y4

a - - - - - - -

2



314

Before Sorting

loo .

h I

Kainlc Acid 1

(59%)

4 I

Sorted-Responders

loo .

Kainic Acid

Sorted-Non-Responders

l”-

Membrane Potential (mV)

[Cytosolic Calcium] (PM)

Maw, Maw, and Barker

100

80-

60-

40-

Acetylcholine

,I

t

, I

1 ,

. i

; :

Fig. 12




315

nonresponding populations after sorting, the cells were rinsed

twice in physiological saline, restained with oxonol and restimu-

lated with 100 IAM kainic acid. Virtually all sorted responders

depolarized again after restimulation, confirming the purity and

functional viability of the sort. By contrast, none of sorted non-

responder cells depolarized to kainic acid after restimulation.

Similarly, acetylcholine restimulation of sorted acetylcholine-

responding and nonresponbding subpopulations revealed >95

purity of each sort. The results confirm that functional sorting

according to both membrane potential and Ca2+c esponses is very

effective, and provides the opportunity for further study of very

specific cell subpopulations in developing CNS.

8. Conclusion

In this chapter, we have described

several strategies for identi-

fying and studying different phenotypic, proliferative, apoptotic,

and physiological properties of developing CNS cells using flow

cytometry. The sort capability of flow cytometers further allows

isolation and purification of subpopulations of CNS cells express-

ing specific epitopes or functional receptors for more detailed cel-

lular and molecular analyses in culture. With these strategies, we

have begun to map the biological properties of CNS cells in the

context of lineage progression. In sum, the versatility, objectivity

and sort capability of flow cytometry may be ideally suited for

confronting the complexity of CNS development, providing an

unparalleled perspective on the distribution of physiologically

relevant properties as the cells transform from proliferative to a

more differentiated state.

Fig. 12. (previous page) Functional sorting of responding and nonre-

sponding cells according to membrane potential and calcium signals.

El9 cells were loaded with either oxonol, a voltage-sensitive dye (panel

A), or Fluo-3, a calcium-sensitive dye (panel B) and sorted into respond-

ing and nonrespondmg populations after the addition of 100 ~JM kainic

acid to oxonol-loaded cells or 10 PM acetylcholine to Fluo-3-loaded cells

(sorting gates are shown as shaded areas). Reanalyses of sorted and

restimulated subpopulations revealed > 95 purity of functionally re-

sponsive and nonresponsive cells.




Bibliography

Abney, E R , Wllhams, B P , and Raff, M C. (1983) Tracmg the development of

olrgodendrocytes from precursor cells using monoclonal antrbodies, fluo-

rescence-activated cell sorting, and cell culture

Dev Bzol

100, 166-171

Altman, J and Bayer, S A 1995) Atlas

of Prenatal Rat Brazn Development,

CRC

Press,Boca Raton, FL

Barry, P H and Lynch, J. W 1991) Liquid Junctron potentials and small cell

effects m patch-clamp analysis [published erratum appearsm ] Membr Blol

1992Feb,125 3)2861 ] Membr Brol 121,101-117

Bear, M F and Malenka, R C 1994)Synaptic plastlclty LTP and LTD Curr

Open Neurobtol 4,389-399

Brent, L. H , Gong, Q , Ross,J M , and Wreland, S J 1993) Mrtogen-activated

Ca++channelsm human B lymphocytes J Cell Physrol 155,520-529

Breuer, W V , Mack, E , and Rothstem, A 1988)Actlvatron of K’ and Cl- chan-

nels by Ca2+ nd cyclrc AMP m dlssocrated kidney eprthehal MDCK) cells

Pflugers Arch

411,450-455

Cruclam, R A , Barker, J L , Zasloff, M., Chen, H C , and Colamomcr, 0 1991)

Antrbrotrc magamins exert cytolytrc actrvrty against transformed cell lines

through channel formatron. Proc Nat1 Acad Sci USA 88,3792-3796

Darzynkrewlcz, Z , Bruno, S , Del Bmo, G , Gorczyca, W , Hotz, M A, Lassota,

P , and Traganos, F 1992) Features of apoptotrc cells measured by flow

cytometry Cytometry 13, 795-808

dr Porzro, U , Smith, S V , Novotny, E A , Morelh, F , and Barker, J L 1993)

Two functronally different glutamate receptors of the kamate subtype m em-

bryonic rat mesencephahccells Exp Neural 120,202-213

Dolbeare, F , Gratzner, H , Pallavrcml, M G , and Gray, J W 1983) Flow

cytometric measurement of total DNA content and mcorporated bromo-

deoxyurrdme

Proc Nat1 Acad Scz USA 80,5573-5577

Flszman, M L , Behar, T , Lange, G D , Smith, S V , Novotny, E A, and Barker,

J L 1993) GABAerglc cells and signals appear together in the early post-

mrtotic period of telencephalrcand strratal development

Brazn Res Dev Brain

Res 73,243-251

Franklin, J L and Johnson, E M 1994) Block of neuronal apoptosts by a sus-

tamed increase of steady-state free Ca2+ oncentration

Phllos Trans Royal

Sot London B Brol Scl 345,251-256

Gallm, W J and Greenberg, M E 1995)Calcium regulation of gene expression

m neurons the mode of entry matters Curr Opm Neurobrol 5,367-374

Gratzner, H G 1982) Monoclonal antibody to 5-bromo- and 5-rododeoxy-

urrdme a new reagent for detection of DNA repllcatlon Scrence 218,

474-475

Grynkrewrcz, G , Poeme, M , and Tslen, R Y 1985) A new generation of Ca2+

mdrcators with greatly improved fluorescence properties ] Blol Chem 260,

3440-3450

Hebel, R and Stromberg, M W 1986)

Anatomy and Embryology @the Laboratory

Rat, BloMed Verlag , Worthsee

Hesketh, T R, Smith, G A, Moore, J P , Taylor, M V, and Metcalfe, J C

1983) Free cytoplasmrc calcium concentratron and the mltogemc strmula-

tron of lymphocytes J Blol Chem 258,4876-4882



CNS Development Studies 317

Hil le, B (1992) lonlc Channels of Excttable Membranes, Smauer Assocrates,

Sunderland, MA

Hockheld, S and McKay, R D. (1985) Identification of major cell classes m the

developing mammalian nervous system J Neuroscr 5,3310-3328

Huettner, J E and Baughman, R W (1986) Primary culture of identified neu-

rons from the visual cortex of postnatal rats. I Neurosct 6,3044-3060.

Johnson, M I and Argiro, V. (1983) Techniques m the tissue culture of rat sym-

pathetic neurons Mefhods Enzymol 103,334-347

Kao, J P , Harootuman, A T , and Tsien, R. Y. (1989) Photochemically gener-

ated cytosollc calcium pulses and therr detection by flue-3 I Brol Chem 264,

8179-8184

Komuro, H and Rakrc, P (1992) Selective role of N-type calcium channels m

neuronal migration. Scrence 257,806-809.

Koopman, G., Reutelmgsperger, C P , Kuilten, G A, Keehnen, R M , Pals, S.

T , and van Oers, M H (1994) Annexm V for flow cytometric detection of

phosphatidylserme expression on B cells undergoing apoptosls Blood 84,

1415-1420

Kostyuk, P and Verkhratsky, A (1994) Calcium stores m neurons and gha

Neuroscience 63,381-404

Koulakoff, A, Bizzmi, B , and Berwald-Netter, Y (1983) Neuronal acqursrtion

of tetanus toxin bmdmg sites relatronship with the last mrtotic cycle Dev

Bzol 100,350-357

Krueger, C., Pull, E , and Kim, S U. (1991) Development of resting membrane

potentials of embryonic murme spinal cord cells evaluated by flow cytometric

analysis Dev Neuroscl 13,11-19

MacDougall, S L , Grmstein, S , and Gelfand, E W (1988) Activation of Ca2+-

dependent K’ channels m human B lymphocytes by anti-immunoglobulin J

Clan lnvest 81,449-454

Mandler, R N., Schaffner, A E , Novotny, E A., Lange, G D , and Barker, J L

(1988) Flow cytometric analysis of membrane potential in embryomc rat spr-

nal cord cells. J Neurosct Meth 22‘203-213.

Marie, D , Marie, I, Ma, W., Lahjoulr, F , Somogyi, R , Wen, X , Sieghart, W ,

Fritschy, J-M, and Barker, J L (1997) Anatomical gradients m proliferation

and differentiation of embryonic rat CNS accessed by buoyant density frac-

tionation a3, 83 and y2 GABA, receptor subunit co-expression by post-mr-

totrc neocortical neurons correlates directly with cell buoyancy Eur J

Neuroscl 9,101-116

Martin, S J , Reutelmgsperger, C P , McGahon, A J , Rader, J A , van Schie, R

C , LaFace, D M , and Green, D R (1995) Early redistribution of plasma

membrane phosphatidylserme IS a general feature of apoptosls regardless

of the imtratmg stimulus mhrbitron by overexpression of Bcl-2 and Abl. J

Exp Med 182,1545-1556

Mattson, M I’ and Kater, S B (1987) Calcium regulation of neurite elongation

and growth cone motility J Neuroscz 7,4034-4043

McCloskey, T W , Oyaizu, N , Coronesl, M., and Pahwa, S (1994) Use of a flow

cytometric assay to quantitate apoptosis in human lymphocytes Clan

Immunol lmmunopathol 71,14-18

Melamed, M. R , Lmdmo, T., and Mendelsohn, M. L. (1990) Flow Cytometry and

Sortrng, Wiley-Liss, NY




Minta, A , Kao, J I-‘, and Tsien, R Y. (1989) Fluorescent mdlcators for cytosolic

calcium based on rhodamme and fluorescem chromophores J Blol Chem

264,8171-8178

Naruse, I. and Keino, H (1995) Apoptosis m the developmg CNS Prog Neurobrol

47,135-155

Petit, J M , Derus-Gay, M , and Ratinaud, M. H (1993) Assessment of fluoro-

chromes for cellular structure and function studies by flow cytometry Blol

Cell 78, 1-13

Raff, M C , Mirsky, R, Frelds, K L , Lisak, R I’, Dorfman, S H , Srlberberg, D

H , Gregson, N A, Leibowitz, S , and Kennedy, M. C. (1978) Galacto-

cerebroslde is a specific cell-surface antigemc marker for ohgodendrocytes

in culture Nature 274,813-816

Schachner, M , Kim, S K , and Zehnle, R (1981) Developmental expression m

central and peripheral nervous system of olrgodendrocyte cell surface

antrgens (0 antigens) recognized by monoclonal antibodies Dev Brol 83,

328-338

Schaffner, A E and Daniels, M P (1982) Conditioned medium from cultures

of embryonic neurons contains a high molecular weight factor which

induces acetylcholme receptor aggregation on cultured myotubes 1

Neurosci 2,623-632

Spitzer, N. C. (1994) Spontaneous Ca*+ spikes and waves in embryonic neurons

slgnalmg systems for differentiation. Trends Neuroscr 17, 115-118

Telford, W. G., King, L. E , and Fraker, P J (1991) Evaluation of glucocorticoid-

induced DNA fragmentation in mouse thymocytes by flow cytometry Cell

Pro14 24,447-459

Tsien, R Y (1980) New calcium indicators and buffers with high selectivity

against magnesium and protons. design, synthesis, and properties of proto-

type structures. Bzochemistry 19,2396-2404

Tsien, R Y. (1989) Fluorescent probes of cell signaling Ann Rev Neurosct 12,

227-253.

Vandenberghe, P. A. and Ceuppens, J. L (1990) Flow cytometric measurement

of cytoplasmic free calcium m human peripheral blood T lymphocytes with

fluo-3, a new fluorescent calcurm indicator J Immunol Meth 127,197-205

Walton, M K., Schaffner, A E , and Barker, J L. (1993) Sodium channels, GABA,

receptors, and glutamate receptors develop sequentrally on embryomc rat

spinal cord cells J. Neuroscl 13,2068-2084

Date post:	18-Feb-2018
Category:	Documents
Upload:	taro
View:	217 times
Download:	0 times

9 Flow Cytometric Strategies to Study CNS Development

Documents