0022-1554/78/2603-01 THE

70$02.00/0

JOURNAl.

Copyright

OF

HISTOCHEMISTHY

© 1978 by The

AND

Histochemical

FLOW-CYTOMETRIC DAVID

BLOCH,

Botany and

Cell

Vol. 26, No. 3, pp. 170-186, 1978 Printed in (ISA.

CYTOCHEMISTRY

Society,

Inc.

ANALYSIS NARLIN

BEATY2,

OF

CHI JAMES

Department and Cell Research Biology Section, National Center

Received

for publication

September

CHICKEN

TSEH FU, L. PIPKIN,

Institute,

RED

EUGENE

BLOOD

CHIN,4

CELLS’

JEAN

SMITH

ANt)

Jit

University

for Toxicological

of Texas

Research,

19, 1977, and in revised

form

at Austin,

Texas

Jefferson, November

78712

Arkansas

72079

14, 1977

Flow-cytometric analysis of acriflavin-Feulgen stained chicken erythrocytes shows a complex distribution of amounts of deoxyribonucleic acid fluorescence, the profile consisting of a main peak and a right hand shoulder. This bimodal distribution, an artifact characteristically seen on analysis of flattened cells using orthogonal flow systems, results from fluorescence emission in preferred directions stemming from the combined effects of refractility and orientation of the cells. The shoulder disappears on analysis of lysed erythrocyte ghosts, also on analysis of cells in a medium whose refractive index approximates that of the cells. An orientation effect for mature erythrocytes was indicated by reanalysis of fractions after sorting on the basis of high and low fluorescence or scatter signals. Both fractions gave the original range of values on reanalysis, although some changes in shape of the profile and in the peak positions for the sorted cells were seen. Sodium dodecyl sulfate treatment of stained cells “loosened” the cells’ structure, yielding lowered scatter values, and fluorescence values approaching those of the shoulder. The average fluorescence emission of the erythrocytes was lower than that of reticulocytes and lymphocytes. The values of the latter correspond closely, although coincidently, to that of the erythrocyte shoulder values. Dual parameter analysis of forward light scatter, and of fluorescence, which is detected at 90#{176} to the laser beam, showed the low fluorescence to be accompanied by low scatter signal, and the high fluorescence among the cells with the high scatter signal. The lowered forward scatter signal is due to a wider scattering of light from cells oriented edge-on to the detector, and loss of signal beyond the acceptance angle of the detector. These results suggest that the preferred directions for fluorescence are in the plane of the cells, and the values are dependent on the cells’ orientation in the stream. These interpretations were supported by the results of analysis of partially oriented cells. The approaches used and conclusions arrived at are similar to those of Gledhill eta! (16), Van Dilla eta! (37), in their analyses of fluorescence of flat sperm cells although the effects in the case of the erythrocytes are less extreme. A number

of reports

alous staining and reticulocytes. 23% drop in the (DNA) staining

have

described

an anom-

behavior of chicken erythrocytes Kernell et al. (19) reported amount of deoxyribonucleic as chicken erythropoietic

progress from cytes. Campbell a

acridine ation

acid cells

to mature erythro(10) found higher

fluorescence and lower in erythroblasts

There

are

also

reports

denaturthan

in

of 3H-thy-

midine incorporation into circulating erythrocytes of chicken (32) and ducks (26). Others have been unable to demonstrate a deviation from DNA constancy in the chick blood line (9, 39, 40). In view of these disparate observations and of the well documented findings of nonmitochondria cytoplasmic (24, 31) we undertook

of Arts.

Present address: Biochemistry Department, igan State University, East Lansing, Michigan. Present address: Department of Zoology, sity of Texas at Austin. 2

orange temperatures

erythrocytes.

‘This work was supported in part by Grants GM09654 from the National Institutes of Health, GS30749 from the National Science Foundation and a Bio-Medical Research Support Grant from the University of Texas. Part of the work was done under tenure of a Research Career Development Award, 5K3GM4253. A portion of the work was done by N. B. in partial fulfillment of the requirements for the degree of Master

erythroblasts and Gledhill

DNA in a few cell types to investigate the relation-

ship between DNA amounts and development during the later stages of erythrocyte differentiation in the chick, using flow-cytometry. These

MichUniver-

methods

also

show

an

170

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anomalous

staining

be-

FCM havior

for

these

from the the basis In

cells,

expected of staining

this

paper

intensity

of

although

values and

we

describe

and

cell orientation.

appeared

Although

to corroborate

DNA changes

differences were

influences and revealed none. due to artifacts of development nomenon al. (16)

the

during found to

in the

from

mature

which we attribute and in the pat-

distributions, of refractility,

which reflect cell shape

an earlier

analysis

existence

of some

development be due to

wEj 500 40C 300

in cell

shape

flow-cytometric staining during

S.,,

200 00 . .‘..m’#{149} 20304050607080

0 I0

.

.

CHANNEL

NUMBER

(3), the the above

parallels that described by Gledhill et for flat sperm and is of general interest

changes

800

80C 600 400 Cl) - -0

LI’

-

uO

aoo

methods to assess

and

‘p0

eoc coo

a search for cytoplasmic DNA The anomalous differences are to which cells in different states are variably subject. The phe-

to those seeking to use to quantitate fluorescence

171

ERYTHROCYTES

departures

differences signals

erythrocytes, in condensation,

terns of fluorescence the combined effects

the

OF

can be explained on instrumental artifact.

fluorescence

and immature to differences

ANALYSIS

700 600

development.

500 400

AND

MATERIALS

300

METHODS

200

Chemicals: Heparin was obtained from Organon under the brand name Liquaemin Sodium “10”. Phenylhydrazine and diphenylamine were obtained from Fisher Scientific Co. Acridine orange and acriflavin hydrochloride were from Allied Chemical. Pancreatic deoxyribonuclease I, grade B, and propidium di-iodide, grade A were purchased from Calbiochem. The National Aniline Division furnished the brilliant cresyl blue, and ribonuclease-A was supplied by Sigma Chemical Company. Erythrocyte

preparations:

White

Leghorn

chick-

ens (strain 96, line WC), and other lines were obtained from Hy-Line International Research Laboratories in Johnston, Iowa. Whole blood was removed from either the heart or wing vein and prevented from clotting with one part Liquaemin to 250 parts whole blood. To 10 parts of chilled blood was then added one part of neutralized formalin, or alternatively, the cells were pelleted by centrifugation at 200 x g for 10 mis, at 4#{176}C, then washed in acid-citrate-dextrose (4). The washed cells were then stored at 4#{176}C or fixed in 10% neutral formalin, or in cold 70% ethanol as indicated below. To obtain a slightly more pure erythrocyte fraction, above

the and

the

whole buffy

blood coat

was was

sometimes removed

pelleted with

as

a syringe.

Reticulocyte preparations: Reticulocytes were obtained by a procedure modified from that of Sanders et al. (30). Chickens, 4 weeks old or more, received intraperitoneal injections once each day for 3 days with 18 mg/kg of 1.8% phenylhydrazine hydrochloride in 0.85% saline. The bird was bled to death 48 hr after

00 0

-

0

,

20

30 CHANNEL

40

50 NUMBER

60

70

80

FIG. 1. Top curves: DNA fluorescence profiles of acriflavin-Feulgen stained cells from a normal (closed circles) and phenylhydrazine treated (open circles) white leghorn chicken. Bottom curves: The same for lymphocytes (open circles) and erythrocytes (closed circles) from another strain. The channel number indicates the relative amount of fluorescence. The shoulder on the right hand side of the main peak belongs to those mature erythrocytes whose orientation is edgeon to the fluorescence detector, and to a lesser extent, to reticulocytes whose orientation doesn’t matter.

the last injection. Reticulocytes were identified by the brilliant cresyl blue method (23) and by the acridine orange technique (1, 38) and percentages were determined with the latter. Fluorescent-Feulgen detection of DNA: The acriflavin-Feulgen procedure is that of Tobey et al. (35) as modified where indicated. The cells were fixed in the cold in 10% neutralized formalin, overnight. These were then rinsed twice in cold water and hydrolyzed at room temperature in 5 N HC1 for 20 mm. Each solution change entailed centrifuging at 1,000 x g for 5 mm. After two rinses with water to remove the HCI, the cells were stained with acriflavine Feulgen for 20 mm at room temperature. The stain was prepared by adding 300 mg of potassium meta-bisulfite and 18 mg of acriflavine hydrochloride to 60 ml of

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BLOCH

172

FIG. immature nuclear

2. Acridine reticulocytes fluorescence

stained blood cells from a white leghorn with their cytoplasmic fluorescence (red), exclusively (yellow). Magnification x650.

repeated

in distilled

twice. water,

Finally examined

HC1, tema 0.2 cells parts step

the

cells

were

suspended

with

the

light

microscope

or fluorescent microscope and measured with the flowcytometer. Deoxyribonuclease (DNAase) hydrolysis was carried out on formalin fixed cells after heating the fixed cells in a boiling water bath for 2 mm (33). DNAase treatment: Cells that were washed in citric acid dextrose (ACD) were rinsed in 0.85% NaCl two times and then suspended in a 37#{176}C incubation medium containing 3 mg/mI DNAase and 5mM MgCl2,

various

pH

7.0.

amounts

AL.

orange

water. To this solution was added 6 ml of 0.5 N and the mixture was allowed to stand at room perature for 5 mm. It was then filtered through z Millipore filter and used. After staining, the were suspended in 1 part concentrated HC1 in 99 70% ethanol and allowed to remain 3 mis. This was

ET

They

remained

of time.

As

in

this

a control,

medium

for

cells

were

and

chicken. Note the mature

the difference erythrocytes,

between the which exhibit

incubated in a similar medium without DNAase. Alternatively, formalin fixed cells were treated with boiling water for 2 mm to release the formaldehyde, then DNAase treated (33). Brffliant cresyl blue staining: The method was that of Magath and Higgins (23), and involves the following: 1% brilliant cresyl blue in 0.85% saline plus 0.2% potassium oxalate was mixed one to one with fresh whole blood for 1 mis, after which a smear was made and examined under the light microscope. Reticulocytes were identified as those cells which had a ring of darkly stained material surrounding the nucleus.

Acridine orange staining: The acridine orange staining procedure was developed for use in this laboratory following the guidelines set out by Armstrong (1) and adapted for identifying reticulocytes as described by Vander et al. (38). A fresh solution of 0.01%

Downloaded from jhc.sagepub.com at UNIV ARIZONA LIBRARY on June 1, 2015

FCM

ANALYSIS

OF

ERYTHROCYTES

173 0

-

1r0

.9Sa

o

a

0

#{176} 0

8(D

.

59 o

o Io

0

8cr. o

8 8

ll

o

0

Z

0_.____._____o

f0

O0

W Z Z

4

o__._.._...___.

-_.....

an n to.

.

‘

S

a n

-c

u

(‘4

§o

Ifi

-O

_______________________ 0 o

o

on a’ 0

-n

2 I

2

I

I

0

04

00

#{231}.OIX ST133

JO

Sn

En

IJ9ViflN

OS

-a n

-

no -an

E-

.

h 5 -I.

no

.5 ?5

On

-J

-il

w

z z 4 I

0.5

-c

.5

‘

nn .O .

n

‘ll no

#{231}..OI ST133

JO

38I’NflN

Downloaded from jhc.sagepub.com at UNIV ARIZONA LIBRARY on June 1, 2015

.t

174

BLOCH

acridine orange in a pH 4, acetate buffer containing 0.85% saline is added to fresh whole blood in the ratio of two parts stain to one part blood. After 20 mm of staining in suspension, the cells were placed wet onto a slide and after 20 to 30 mm, counts were made. Reticulocytes were identified as those cells which had an orange-red cytoplasm, as seen with a fluorescence microscope. Fluorescence microscopy: Fluorescence microscopy was done using an Osram HBO 200W mercury vapor lamp focused with a dark field condenser and fitted with exciter filters BG12 and UG2 to pass light in the range 330 to 400 nm. Yellow and green barrier filters

were

is

eliminated

(except

for

Raman

scatter)

by

the use of a 515 nm yellow barrier filter. The primary component of the system is a flow cell and detector unit manufactured by Research Developments, Inc., Los Alamos, N.M. Flow is maintained at a steady rate with a Millipore vacuum pump at a pressure of 0.867 atmospheres and with sheath and stream

reservoir

levels

consistent

with

maintaining

a

narrow stream diameter. The light source is a 52G Argon Ion Laser from Coherent Radiation in Palo Alto, Calif. This device will produce a continuous exciting beam at 488 nm with a constant power output of 300 milliwatts. Pulses

from

an NS-621 Series

the

Memory Display

on

a cathode

ferred

photomultiplier

Analog

Inc.

Digital

Unit by

produced

the

ray

to printed

are

Converter

tape

unit

and

the

using

by

0 S

ci) .J

-J w C-)

Iii

z

Scientific,

X-Y

is a direct

plot

information

is trans-

a Hewlett-Packard

digital

Light

stained arrow

4.

at

the photomultiplier filter,

90#{176} was

tubes

filter.

A barrier detecting

was

using

of the detector

or alternatively

scatter

measured with

superimposed

photomultiplier

the

using

Fluorescence

a Becton-Dickinson

two

of

a

ces

of the

of the

parameter

scattering and fluoresanalyses were carried out Activated

Cell

II).

facilitated

by

ualization

of the

use

of three

the

Program dimensional

“Perspec” representation

for

mixtures

the

visof

the two dimensional profile. This program is based on the methods of Szabro and Guiieri (34) as modified by Phillips (27). Matching the refractive index of the cells: The

entire

with

were

fluid

CONTENT

of acriflavine-Feulgen lysed erythrocytes.

determined

system

70% ethanol,

appropriate

The

for intact

using

aniline-ethylene

in the and

cells.

a Bausch

flow-cytometer

this substituted glycol mixture.

was with

the

of cells: Cells were oriented by inin the nozzle assembly of the FACS H that was beveled at 45#{176} from the vertical, on each side, such that the angle of the tip of the tube was 90#{176}. The tube is rotatable so that any orientation around the cells long axis can be obtained. This approach was suggested by Stovell,4 Fulwyler5 and has recently been described (14, 15). Cell counts for quantitative DNA assays: Whole blood samples which had been pelleted, and the buffy coat removed, were suspended in ACD and coded. During the counting procedure the suspension was maintained with constant stirring in a beaker and samples to be counted were removed from different Orientation

serting

green

Low angle light scatter measurements were done using an open diaphragm, giving an acceptance angle of from 0.5 to about 15#{176}. Interpretation of the results of the two parameter analysis was (FACS

DNA profile

refractiity of the cells was decreased by suspending them in a medium whose refractive index approximated that of the cell. Mixtures of freshly distilled aniline and ethylene glycol were made. Cells dehydrated with alcohol were suspended in these mixtures and examined with a phase microscope, using a yellow filter in order to diminish the visibility of the acriflavine. The best refractive index match was considered to be that which gave the least contrast when the cells were examined with phase. The nucleus and cytoplasm could not both be matched with the same mixture, so a compromise was made, selecting the mixture that gave the least “overall” contrast. The refractive indi-

ysis,

with

band

in front in

analysis to help balance cence signals. Alternatively,

unit,

a broad

one

Fluorescence

nuclei obtained from shows the position of the peak

rinsed scattering

nm

RELATIVE FIG.

and Lomb refractometer. Useful values ranged between 1.500-1.550. These high refractive indices are characteristic of formalin-fixed cells that are examined under conditions of extreme dehydration. Before anal-

an NS-636

by Northern

memory tube,

processed

and

recorder.

Sorter

N

cytometry

scatter

480

AL.

used.

and cell sorting: The flow cytometer as used here is described in several papers (Kraemer et a!. (20), Holm and Cram (17), Van Dilla et a!. (37)) The optical unit of our instrument contains an RCA Development Type C7164R Photomultiplier Tube with a spectral range from 400 to 900 nm. Light Flow

ET

a tube

4Stovell 5Fulwyler

R: Manuscript MJ: Personal

Downloaded from jhc.sagepub.com at UNIV ARIZONA LIBRARY on June 1, 2015

in preparation. communication.

FCM

ANALYSIS

OF

ERYTHROCYTES

175

as described above. When used as a starting material for isolation of DNA the nuclei were pelleted by centrifugation at 1,000 x g. Purification of DNA: Hydroxyapatite (HAP) was used according to Britten et a!. (7) for isolation, purification and concentration of DNA, using isolated nuclei as starting material, and also the supernatant obtained after pelleting the nuclei. Typically, 4 g of HAP (Bio-Gel, HTP, Bio-Rad Laboratories, Richmond, Calif.) were suspended in 8 M urea, 0.24 M sodium phosphate (equimolar monoand di-sodium) and washed several times in this mixture. Fractions

depths and positions in the beaker. Twenty different counts were made on each sample using a Bright-Line hemocytometer produced by American Optical. Alternatively, counts were made using a model ZR Coulter Counter. Isolation of nuclei: Cells that had been washed in ACD were diluted 100:1 with 0.1 x saline sodium citrate (SSC) containing 5 mM EDTA. This solution gently lysed the cells, so that when centrifuged at 150 x g for 5 mis, the nuclei, or erythrocyte ghosts, pelleted. When used for flow cytometric analysis, the pellet was dispersed in the above medium, fixed and

stained

400 o9o.

0

300 0 0

200

50

00 LOW

ANGLE

50

SCATTER

600

0

00

-J -J

0

w

0

00

0

0

000

400

0 0

w

0

z

0

#{149}

0

S

200 #{149}

#{149}S#{149}

000

0

0

05

UNSORTED

00

#{149}

0 OS

#{149}

% o0

S

00

#{149}

#{149}

S -

S

#{149}

#{149}#{149}

0 0

#{149}

0

#{149}

LH

0

#{149}‘

0

0

0 0

0

#{176}‘b

00

op

20

40

60

80

FLUORESCENCE

FIG. 5. Fluorescence profiles of subpopulations obtained from mature cells hand peak and from the right-hand peak of the scatter profile (shaded portions). range of fluorescence values in both fractions.

by sorting cells from Note representation

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the leftof entire

176

BLOCH

ET

AL.

600 00

&

500

0

400 b

0

#{176}

& 300

0

#{176}o:, p

200 ci) -J .J

0

81

00

w

0

0

0

0-I

0

500

0

8

cci 400

0 0

60

0 000

00

300

o0

60 0

&

0

200

0 0

0

00

0&

0

too

50 CHANNEL

FIG. number.

6. Forward Abscissa,

obtained

after

buffer

and

HAP. The the eluate then

scatter intensity

column was free

was washed of material

with the absorbing

with

24 mM

carried

out

then

0.40

DNA,

both

at room of

glycerol,

buffer,

pH

were 0.10

-40#{176}C.6 The

mersed

in

monitored dients,

The

chamber by

sedimentation

its

DNA,

The

from

an

was

imwas

in sucrose RNA was

denatured

by

heating

reannealed

by

incubating

hydroxyapatite

columns

held

the

RI:

Personal

communication.

or

of Davies

sodium

measured

varying

from

Dimensions micrometer. the

calculated

were Cell

at 60#{176}C.

(8),

(SDS)

smaller

nuclear

solution,

and correspond-

taken thickness

using was

a caliesti-

Mass

diameter.

was

as follows: Mismass

4, is retardation where

4,

=

A

=

2a A/360-

X

irab

a

A is area a is angle between identical color transitions of background and object A is wavelength, 551 rim. X

is specific crement,

of

refractive of 0.18

a is #{189} the minor

(7). Reannealstranded

sulfate

a X 40 objective

at

lengths

Burton

Dry mass determinainterference microscope et a!. (13). Erythrocytes

dodecyl

using

ing condenser. brated ocular

b is #{189} the major

DNA Readings central

6Bntten

method

water,

stan-

in

diphenyla-

to

at 60#{176}C for 90 mis.

concentrated

and

salt concentrations assay for double

cells

The

according

cell

or on hydrolyzed DNA by hydrolysis in 5 M perchloric

HAP.

then

by

from

DNA:

Ordinate:

cells,

M=%4,A

graas

was

for

at

size

bath

time in appropriate ing was followed

washed

mated

hour

DNA

concentrations

in

The

transfer

shearing, eluting

400 DNA

water

60#{176}C at various

using

following

on whole,

were

purified

sample

bath. rate

and

on and

half

the

dry-ice

ribosomal

either

curve).

for

used

using

M phosphate

0.12 for

containing

ethanol

was

stranded

of the

acetate,

test

procedure

extracted

(bottom

Dry mass determinations: tions were made with a Zeiss

to approximately

blendor

a 70%

by readsorbing boiling

Msodium

cells

stranded

samples

a solution

in a Virtis

using

dards.

sheared

mature

mine

in DNA

by blending

7.0,

double

and

acid

buffer,

single

remove

reannealing:

pairs

and

200

Diphenylamine

Fractional

M phosphate

0.12

curve)

this

buffer until at 260 nm,

buffer.

(RNA) to

in prewashed

temperature.

on hydroxyapatite, 67%

with

M buffer

DNA,

Kinetics

phosphate

acid

(top

suspended of packed,

ribonucleic

nucleotide

were

to a column

was

to remove

incubation

applied

washed

elution

profiles of reticulocytes of scatter signal.

ISO NUMBER

cytoplasm

for plug

the

nucleus

through

were

taken

the

were

through

Downloaded from jhc.sagepub.com at UNIV ARIZONA LIBRARY on June 1, 2015

taken

using

Readings a similar plug

nucleus.

incm3

axis, axis

g’

and

a small

for the adjacent

FCM to

the

nucleus.

ellipsoidal

The

shape,

calculations

which

the

ANALYSIS

assumed

erythrocyte

OF

DNAase

a regular approximates.

1 shows

fluorescence

the

chicken.

of amounts

acriflavin-Feulgen

red blood A main peak

cells and

main peak Note that

are the

typically shoulder

of

G-2

and

does

presence

value,

of S period

4C position is due of pairs of adherent sional tions

smaller are

decreased

is unaffected

by

cells.

not

The

at the

in these far short the

peak

at the

in the sample peak and occa-

6C and

higher The

treated shown).

with Lack

posi-

shoulder

treatment.

The fluorescent staining is dependent hydrolysis. The fluorescence of both peak and shoulder declines when DNAase of residual

on acid the main cells are

before staining fluorescence

iSA

bility

of

for

(not in the

DNA

the

staining

represent

small

by sonication. this

stained

seen falls

to the presence cells. This

peaks

of

from a white leghorn a shoulder to the right

of the samples. the

samples

responsible

indicated

the

DNAase sensitive. Since DNA might be attributable

distribution

among

peripheral

treated

terial

RESULTS Figure

177

ERYTHROCYTES

reaction,

hydrolysis lyzed for hours. creased

with

45 mm,

followed

distribution (not

times

The peak

maximum remained

which

orientation

an overall

reflects of the

decrease

sta-

of the

regard

to acid

between The the

difference

in-

shape

of the however

in fluorescence

be-

to altered DNA hy-

of the erythrocytes One is an anomalous

cytoplasmic cells, in fluorescence

flattened

several 10 and

same

and shoulder is not due of some of the cells to

drolysis. The fluorescent staining influenced by two effects. signal

with

by a decline.

profile

differential

conditions

from 1 mm to of the fluorescence

a broad

shown).

tween the succeptibiity

of

of a sample were hydrowith 4 N HC1 at room

ranging intensity

The

is

staining

the

aliquots

various

temperature,

ma-

differential

particularly

(29),

the

staining

to

under

that

incremental

is

refractiity and and the other, which is char-

FLUOS.EScENCE

FIG. 7. Three dimensional display of the results and DNA fluorescence (x-axis) of mature erythrocytes. cells. The light scatter is a function of cell size and contour lines as viewed from the top.

of dual The orientation.

parameter analysis of forward light scatter (y-axis) z-axis shows cell number. Acriflavin-Feulgen stained The insert, taken from the same data, shows the

Downloaded from jhc.sagepub.com at UNIV ARIZONA LIBRARY on June 1, 2015

178

BLOCH

acteristic due

of the

erythrocyte

to condensation The decreased

by

and

of the fluorescent

comparison

of

the

presumed

nucleus. staining

is shown

fluorescence

of

erythrocytes and of reticulocytes cytes, neither of the latter showing lous effects exhibited by the mature 1). Strain

96 line

WC

number of circulating prise up to 15% of the lation part

(Fig. of the

enriched

lighter

be to

has

2). These early cells right hand shoulder. of

obtained settle,

layers.

by taking

allowing

Alternatively,

The

bimodal

The

compopu-

fixed

a

the

of upper,

reticulo-

of mature

tectors.

The

effect

of refractiity

the loss of the shoulder in a mixture of ethylene

Fi;. is the

cells

of cytoplasmic relative to the is indicated

when whole cells are glvcol and aniline,

8. The same as Figure 7, except same as was used for Figure 3.

that

is redeby run of

90#{176} scatter

and

of

1.500 This

main

fect

contribution is shown by

cells and

are lysed before nuclei of ghosts

than

the

of

the

the

manner

a merging

(Fig.

3).

cells are reappears.

The

brought This

by Gledhill sperm cells.

et al.

of of the

effect

is

back effect

to is

in their

of the cytoplasm to the efloss of the shoulder when fixation. obtained

highly compacted and show an even that

in

causes

peak

When the shoulder

to that noted of flattened

become staining,

cells

suspensions from

enriched

distribution

et al. (16).

similar analysis

cyte populations could be obtained by treatment of chickens with phenylhydrazine, in which case the peak near the position of the shoulder becomes the main peak, all cells having the higher values (Fig. 1). due to the combined effects fractiity and cell orientation

index

Gledhill

reversible. water, the

account for Reticulocyte-.

previously

samples

refractive

a substantial that cell

AL.

shoulder

mature

or lymphothe anomacells (Fig.

reticulocytes total red blood

preparations

could cells

chicken

to be

ET

mature

Isolated in this

during lowered

nuclei manner

fixation and fluorescence

erythrocytes.

In

any

event, the fluorescence values show a near normal distribution (Fig. 4). That orientation, rather than inherent differences among the cells, is the cause of most of the differences between the peak and shoulder when mature cells are analyzed, is shown from the results of isolation and reanalysis of cells giving peak and shoulder signals. Sorting the cells on the basis of high values, or high and low reanalysis of the fractions, spectrum

reanalysis

was

measured

of values. Figure of the fluorescence

instead

of

Downloaded from jhc.sagepub.com at UNIV ARIZONA LIBRARY on June 1, 2015

and low scatter gives

fluorescence values, then the original

5 shows the results of profile of fractions

low angle

scatter.

The

sample

FCM

ANALYSIS

OF

ERYTHROCYTES

179

#{176} .4

*

b

-

-I,)

V k... r

FI;. obtained signals.

on (The

related-see spectrum redistribution pend

upon

9. Comparison

of untreated

the basis of high and scatter and fluorescence

low

Fig.

the

7). In each

fraction,

‘.-

and

presence

of reticulocytes

SI)S

scatter are cororiginal

of values is seen, although there of the frequencies that may the

A

is a de-

in the

#{149}-

treated

erythrocytes.

population. scattering rescence

The cells values.

Comparison properties shows

x22(5)

Magnification

of

differences

modes giving

are

different,

slightly

of the forward erythrocytes in their

Downloaded from jhc.sagepub.com at UNIV ARIZONA LIBRARY on June 1, 2015

the

higher light and

profiles

higher

peak

fluo-

scattering reticulocytes

(Fig.

6). Scat-

180

BLOCH

tering

of

reticulocytes

treated chickens est signals. Scatter has fluorescence. cence

and

signals signals,

from

shows an Dual

parameter

analysis shows

to be accompanied high fluorescence

similarly,

high

fluorescence. show

orescence

FIG.

represent

low

with with

the

the and

distribution

(Fig.

almost 7).

as Figure doublets

entire

scatter

signal

is the

result

of cells

with

the scatter detector’s collecting lens. Decreasing the opening of the iris diaphragm in the scatter detector from 150 decreases the intensity of the

scatter

range

lower

oriented in such a way light at angles ranging

is associsignals, and

higher

AL.

low-

of fluores-

the

fluorescence of scatter

a unimodal

10. The same cells, usually

the

by low fluorescence signals to go with high

low range

scatter, Cells

giving interaction

scatter

forward

The

phenyihydrazine cells

interesting

scatter signals. The ated with the entire

nals

few

ET

of

as to spray the scattered beyond the periphery

of

lower scatter signal before it decreases the higher signal (data not shown). So in this analysis the lower scatter values actually belong to cells with greater scattering properties. Ninety

degree

scatter

sig-

tion

Gaussian

flu-

stretching over (Fig. 8), although

7, except that cells were run and triplets, whose high scatter

with

scatter

fluorescence, the

shows both

no such

interac-

fluorescence

signals

entire range of scatter values there is a higher proportion of

in 1% SDS. The peaks high along values throw them off the scale.

Downloaded from jhc.sagepub.com at UNIV ARIZONA LIBRARY on June 1, 2015

the

y-axis

FCM cells

with

the

cells

the

higher

giving

fluorescent

higher

scatter

orescence.

signal in

lower

and

value

of the

main

peak The

in size,

dry

mass

and

concentration

upward as the shoulder declined. fluorescence is higher and the distoward normality, of emission. The

dramatic swelling of the their normal volume

flu-

fluorescence

TABLE Change

shifted average

tribution tends of directionality

a

increasing

181

ERYTHROCYTES

among

SDS had fluorescence,

and

values

The

OF

scatter.

Running the stained cells marked effect both on scatter shifting

ANALYSIS

of dry

suggesting loss SDS caused a

cells to around without any

8 times obvious

I of erythrocytes

mass

after

treatment

with

1% SDS Overall

Nuclear

Mass

in

Control SDS treated Treated as percentage

120.4 ± 14.9 1023 ± 58 850%

of control

± 2.2

26.7

8.6 ± 1.4 14.8 ± 4.9 172%

39.6 ± 2.9 148%

Con-

Cell Mass

35.3 54.5 154%

g/cm1

0.293 0.053 18%

00

3O0

0

0 0

UNORIE

200

0

N TED

0 0 #{149} 00000000

#{149}#{149} 0 0

100

#{149} 0

0

#{149}

0 #{149}

0 00

0

.1

$

(I)

400-

w

FACE

C-.)

TO #{149}

FLUORESCENCE

#{149}#{149}

#{149}#{149} #{149}#{149}#{149}

DETECTOR 00

#{149} 00 #{149}

0

#{149} 0

0

#{149}

#{149}

0

00

#{149}.

#{149}

.#{149}#{149}#{149}.#{149}

0 00 0

0

600

0

EDGE

TO

0

FLUORESCENCE DETECTOR

400

0

0

0

0 0

0

0 0

0

0

-

T-------

0

FIG.

control.

Fluorescence Middle curve,

11.

face

$

0 I

20 30 FLUORESCENCE

profiles of partially oriented acriflavin-Feulgen to fluorescence detector. Bottom curve, edge

1

40

0

50

stained erythrocytes. to fluorescence detector.

Downloaded from jhc.sagepub.com at UNIV ARIZONA LIBRARY on June 1, 2015

Upper

curve,

BLOCH

182 change eter ing

in shape analysis cells to

scatter

however

(Fig.

now shows belong to

values

(Fig.

the the

10). The

9). Dual

ing the

cells made

with on

ment. Table in a volume

SDS, the

I shows increase

is accompanied 54%. The lowered change these

in the conditions,

entation and Identification

by

dry

cells,

the

tube

orientation

was

only

cells

clear,

and

effect scatter signal.

that limiting the acceptance detector has on changing

appeared

mass

that the of more mass

to be

treatment. to load-

determinations and

after

increase of only results in a drastic and between

under ori-

the

the

as

conclusions

based angle the

detector, by bevel-

sample

of the scatter

Acnflavin-Feulgen

bear-

stained

mature

chicken

exhibit two anomalous effects on flow-cytometnc analysis. One is a depressed fluorescent staining. The other is a nonrandom radiation of scatter and fluorescence signals. The erythrocytes

is thought to be due primarily of the nucleus and the second

bined effects of cell entation in the flow

refractiity, stream. The

what

end

related

in their

effects

to compacto the comshape and oritwo are some-

but

their

mech-

0

oocP 0

00 0

5OtY UNORIE

NT ED

0 00

0 0

8

0

0 0

0 00 0

0 0

0500

EDGETO SCATTER

U, -J -J

#{149}#{149} DETECTOR #{149}

w 0

I

LL. 0

0%

lb

cc Ui

00

I; 00 0

z

FACE

TO

%0

500 SCATTER

0

0

0 0

0

0

0

DETECTOR

& 00

0

00,

0

I#{212}#{248}

50

LOW ANGLE FIG.

12.

Low angle

scatter

profiles

of partially

is

on the

DISCUSSION

first tion

intensity of signal are altered. of signals with orientation:

to introduce

support

partial,

12, but orientation’s and scatter signal

treat-

swelling, resulting than eight times,

scattering properties, the relationships

used

The

by Figures 11 and on both fluorescence

Cells were oriented edge toward scatter and edge toward fluorescence detector, ing

stream.

shown effect

before

a dry refractiity

ing

param-

AL.

few higher fluorescgroups giving low

much less refractile following SDS Because swelling might be attributable were

ET

oriented

SCATTER

erythrocytes.

Conditions

11.

Downloaded from jhc.sagepub.com at UNIV ARIZONA LIBRARY on June 1, 2015

identical

to those

of Figure

FCM amsms

are

different

rately. Effects

is due

to the

fluorescence to compaction

lowered

mammalian

lowering

dye

sperm

The

swelling

in

of the

binding

and

cells

(16,

36).

isolated nuclei whose collapse

increase

stained

The

signal

cells,

in SDS

shows the effect of compaction A similar effect of SDS on other

also

seen

as an

and aid

systems

this

type

the

the anomalous butions. The

fluorescence effect of the loss

may

resolution

by

of acriflavine-Feulgen

refractility: is a major

by the

on cells

of treatment

in increasing

in analysis

stained DNA. Effects of the cytoplasm

indicated

to

and in is very

in fluorescence

of previously

solutions, quenching. serve

sepa-

and has been attributed cells (10, 19) and in highly

is exaggerated of lysed ghosts

pronounced.

flow

The

quenching, in these

flattened

was

be discussed

OF

staining of the mature cells to about of the reticulocytes and lymphocytes

both

effect nuclei

will

of compaction:

fluorescent 80% of that

on

and

ANALYSIS

The refractility contributing factor and scatter refractive

or lowering

of to

distriindex is

of the

ERYTHROCYTES arated on the basis of high or scatter signals. The number of orientations flow

systems

that

elongated

shear

forces

is limited. with

Refractive

index

and

error in flow-cytometry microspectrophotometry fact

that

advantages, Effects

this

The

by the

of the

compaction have their (28) and

new

importance

loss

technique,

as sources

of in the

all

errors. and

axes

found

oriented parallel

by to the

each

other is face-on

to the fluores-

other face-on These ori-

entations

FSf

are

designated

FtSe

11, the capital letters Fluorescence detectors, noting

edge

and

face.

and

indicating and the Because

in Figure

Scatter subscripts of the

and de-

existence

of intermediate orientations and the finite ertures of the detectors (subtending angles about 90#{176} and 30#{176}, respectively, for fluorescence and scatter) the terms “edge-on” and represent a range of orientations. The existence of preferred directions channeling

and

cells’ orientations is indicated by

forward

scatter,

apof

“face-on” for fluorelative

and the system’s symthe interaction between

PETECTOR

shoul-

precedents point up with

has its own inherent of shape, orientation

of

long

in the

has

cence, edge-on to scatter and the to scatter, edge-on to fluorescence.

FLUORESCENCE

in SDS.

is shown of the cells.

highly

cells

and to

or by swelling

them

(18)

optic axes at 90#{176} to stream). One orientation

to the metry,

cytoplasm on lysis

fluorescence

of the Kachel

become their

low

orientawith regard with their

cence shoulder when the refractive indices of the cell and suspending medium are matched either by running the cells in a nonaqueous medium, the der

cells

and

direction of flow. Thus the erythrocyte’s tions range between two extremes to the detectors (which are mounted

rescence

fluores-

183

of

its

refrac-

tility:

These three factors need to be considered together. Nonspherical shape makes orientation have meaning, but without a refractive index differential, important. SDS treated

shape The

mature untreated a normal or near gests the existance shape effects

and are

and

orientation

reticulocytes erythrocytes

red blood cells, normal distribution. of threshhold

refractiity too small

become

below to be

each

exhibits This sugconditions of

which detected,

orientation and the

discussion immediately following will apply only to the mature erythrocytes, which do show these effects. That difference in cell orientation is the priof the

complex

-

un-

LASER

BEAM

and the enlarged are flat. But unlike

mary

cause

sured values

by the recovery of the entire on reanalysis of cells that had

distributions

is asrange of been sep-

FLOW

13. Schematic depicting orientation of cells presenting opposing profiles (edge or face) to the two detectors that are disposed at 900 to each other and to FIG.

the

stream.

The

vertical

arrow

indicates

direction

of

flow. The cell labeled F1S, face to fluorescence detector, edge to scatter detector, gives the low scatter signal (even though widest scatter-see text) and low fluorescence signal. The cell labeled FeSf, edge to fluorescence, face to scatter, gives the high fluorescence and scatter signals. The Ff S1. orientation is represented by the main peak in the middle curves of Figures 11 and 12, and the F,81 orientation, the main peak in the lower curves of these figures.

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184

BLOCH

_________#{149}

ET

AL.

I

20 V

a,

40 a) C C

0

0

a

60

80

io2

o0

I0_

to2

10’ COT

FIG. 14. Kinetics of reannealing of DNA obtained from chicken nuclei, and from the “cytoplasmic” supernatant of red blood cell lysates. 0, DNA from red blood cell nuclei of WC 96 chickens;#{149}, DNA from red blood cell nuclei of barnyard fowl of unknown genetic origin; 0, DNA from liver nuclei; L, DNA from the cytoplasmic supernatant fraction of lysed red blood cells, from a phenythydrazine treated WC 96 chicken.

the

intensities

of the

eter analysis. accompanied

two

signals

in two-param-

Since low fluorescence by low scatter signals,

fluorescence

by high

the highest signals another, e.g., from

scatter, are edge

signals and

flat

cause

mentioned contributions

existence

of threshhold

above, and by the that reflection

are

sperm,

nuclear

source

negligible. slightly

In the flattened

toplasm, sperm,

the

from

the

piping light and orientation of the and cell

low signals. An enhanced has

been

known

capacity

fluorescence reported

in the

erythrocyte, the prolate spheroid,

which is chromatin

appreciable, is highly

relative refraction

scatter detectors

cells

for

effects of of high

plane et al.

their face orientation

for

by

the In

permitted position

of signal with gain

in one direction in another. The

hand,

according presenting

detector

systems

that

to angle and azimuth their edge to the laser

angle scattering.7 by showing that Loken

MR:

the

by two showing would correlations

inferences to be made about of reflecting surfaces toward hence cellular orientation.

detectors,

signal of cells pre-

Identification correlating

signals measured independently arrayed at different angles,

resolve

the the On

be

distwo the

signals

show the cells to give a wider

This effect could be observed narrowing the acceptance angle

of the (16)

nucleus is a and the cy-

is granular and relathe phenomena response of both cells

to the detector. was done by

micro-

the

cytoplasm

is flat. In condensed.

senting of the

of flat

by Gledhill

the

conditions

phase

from the observed cells on the frequencies

the

and

forward scattering is generated

other

scope,

the

In flat

to changes in refractive index suggests that the basis for explanation should be similar. Regarding scatter: Loken et al. (22) has found

differently

under

different. is very

the blood cell, the nucleus tively diffuse. Nevertheless seem to be similar and the

that loss correlated

cells

very

that the higher chicken erythrocytes

changing and

fluo-

rocytes

make to scatter at varying angles of deflection of the light (11). An assignment can be made, based on the observed effects of limiting the scatter acceptance angle on the scatter signal, from the visual observation of the contrast of oriented

emitted

that

at 90#{176} to one Recalling that

of specific cell orientations to sigbased on theory, is difficult be-

of the

in which

effect from and eryth-

the low scatter signal is due to deflection of much of the light out of the collection angle of the detector, the lower scatter signal is due to a wider angle of scatter. So low fluorescence is accompanied by a wider scatter, high fluorescence by a narrow scatter angle. Since any given cell presents different profiles to the two detectors, edge and face, the greater signal for both fluorescence and scatter are associated with one cellular orientation in the instrument. Assignment nal intensities,

sperm,

rescence is channeled by a “piping” the edges of the sperm. The sperm

it is concluded

generated and face.

mammalian

are high

Personal

Downloaded from jhc.sagepub.com at UNIV ARIZONA LIBRARY on June 1, 2015

communication.

FCM decreased

the

lower

signal,

not

and is evident on examination on in the phase microscope.

ANALYSIS

the

higher

one,

of the

cells

edge-

shifting frequencies low signals permitted with quality tainty. These Figure

by others4 did take

(14). place,

of cells giving assignment

of signal with interpretations

Still, and

apthe

the high and of orientation

a fair degree of cerare summarized in

13.

Fluorescence signal as an indication of cell maturity: The similar values of the reticulocyte peak and the erythrocyte shoulder are thought to be fortuitous. In many erythrocyte preparations, especially from chicken strains whose

blood

circulating

contains

appreciable

reticulocytes,

the

numbers

shoulder

sist of signals from mature cells detector edge-on, and reticulocytes entation doesn’t matter. ered value, whatever the evidence ferentiating separation and late

preparations

Lack We

that

will

con-

pass whose

the ori-

In any event the lowcause, can be taken as

with

of evidence

had

previously

fluorescence

SDS.

for

as indicating

the mature and pretation resulted reinforced ena noted,

cytoplasmic

interpreted

differences

immature cells from fortuitous

one another, including the

DNA:

differences

in the in DNA

(3). This artifacts

of

interthat

one being the phenomlowered fluorescence

and from revealed

the

two

fractions

prevalence such

cytoplasmic would have cytoplasmic with

(Fig.

of DNA

as were

noted

the cytoplasmic no qualitative

in the

the view

fractions differences

14).

of

of different

observed

DNA fractions suggested the DNA fraction, that

fraction tion by

results DNA

from from

menting cytoplasmic

nuclei. The fractions

the

DNA this lysed,

classes

nuclear

are

in the fraction’s broken

observation of lysed

duck

made,

lack

system Graphic

using

labelling

of evidence

for

has been noted representation

surements

is also

techniques

amplification

(5). oftwo

in

parameter

of the three-dimensional of two parameter noteworthy.

mea-

Computer

based

programs for plotting data with different perspectives aid in visualizing the results of complicated two dimensional histograms where multiple

peaks

exist.

The

into a format for a simple matter. have

been

made

application as they

(2)

seem might

transcription

of the

use in computerized Similar uses of this but

its

value

and

not to be as widely

data

plotting approach

is

ease

of

appreciated

be. ACKNOWLEDGMENT

The Sandy

authors

thank

Ewald

for

Dr.

their

Bob help

Sanders and

and

advice

use of the chickens and preparations used in these experiments and

Mr.

Miss in the

of the cells Reuben

Mitchell for his help in the use of the Perspec program for plotting the results of two parameter analysis. The authors also express our appreciation to Drs. Barton Gledhill and Mortimer Mendelsohn of the Lawrence Livermore Laboratory, Dr. Paul and Dr. Michael

Latimer Loken

helpful conversations tion of the results. gift of samples provided by

Mr.

that The from R.

Research authors also

of the Microbiology use of his Coulter to

Kathryn

Clark for their the two-parameter Sternberg for

of Auburn of Stanford,

led to the interpretaauthors appreciate the

several lines B. Arvidson

of chickens, of Hy-Line

Laboratories, Johnston, thank Dr. James Walker

Department Counter. Thanks

Bishop,

University for their

James

Clark

here are and

assistance in compiling analyses and Mr. his help in the contour

for also

the due

Devin data for Michael plotting

programs.

the and

of lymphocytes (24) existence of a real but the similarities case

the

in

repetitive

between

erythrocyte

of lysed between

Differences

been

The

International Iowa. The

the isolated nuclei, the second being a relative ease of extractability of DNA from reticulocyte nuclei under the conditions used for analysis. Reannealing kinetics of DNA obtained from nuclei, cells

also

date: The versatility graphical representation

of

of the maturity of the cell, where difsystems are studied. More complete of the fluorescence signals of the early stages can be effected by treating the

stained

had (25).

of the cells incomplete. tip was less

185

ERYTHROCYTES

this

In our experiments the orientation in the flattened sample stream was The 90#{176} angle of the sample tube acute than that used preciable orientation

OF

compatible cytoplasmic contaminaor nonsedi-

of DNA in the erythrocytes

REFERENCES 1. Armstrong JA: Histochemical differentiation of nucleic acid by means of induced fluorescence. Exp Cell Res 11:640, 1966 2. Arndt-Jovin DJ, Ostertag W, Elsen H, Klimek F, Jovin TM: Studies of cellular differentiation by automated cell separation. Two model systems: Friend virus transformed cells and Hydra attenuata. J Histochem Cytochem 24:332, 1976 3. Beaty N, Bloch DP: Extra DNA during erythrocyte development in a strain of chicken. J Cell Biol 63:18a, 1974 (Abst.)

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BLOCH

186

4. Beutler E: Red Cell Metabolism. New York, Grune and Stratton, 1971 5. Bishop JO, Pemberton R, Baglioni C: Reiteration frequency of haemoglobin genes in the duck. Nature New Biol 235:231, 1972 6. Bloch DP, Chin F, Fu CT: Flow microfluorometric analysis of chromatin, Methods in Cell Biology, Edited by G Stein, J Stein, L Kleinsmith. In press. 7. Britten RJ, Graham DE, Neufeld BR: Analysis of repeating DNA sequences by reassociation, Methods in Enzymology. Edited by L Grossman, K Moldave. 29:363, 1974 8. Burton K: A study of the conditions and mechanism of the diphenylamine test for the colorimetric estimation of deoxyribonucleic acid. Biochem J 62:315, 1956 9. Cameron IL, Prescott DM: RNA and protein metabolism in the maturation of the nucleated chicken erythrocyte. Exp Cell Res 30:609, 1963 10. Campbell GL, Gledhill BL: Chromatin of primitive erythroid cells from the chick embryo. Chromosoma 41:385, 1973 11. Crissman, HA, Mullaney, PF, Steinkamp JA: Methods and Application of flow systems. Meth Cell Biol 9:179, 1975 12. Coates V, March BE: Reticulocyte Counts in the Chicken. Poultry Sci 45:1302, 1966 13. Davies HG, Wilkins MHF, Chayen J, LaCour LF: The use of the interference microscope to determine dry mass in living cells and as a quantitative cytochemical method. Quart J Micro Sci 95:271, 1954 14. Dean PN, Pinkel D, Mendelsohn ML: Simple hydrodynamic orientation of sperm for flow-fluorimetry of DNA. In press. 15. Fulwyler MJ: Hydrodynamic orientation of cells. J Histochem Cytochem 25:781, 1977 16. Gledhill BL, Lake 5, Steinmetz LL, Gray JW, Crawford JR, Dean PN, Van Dilla MA: Flowmicrofluorometric analysis of sperm DNA content: effect of cell shape on the fluorescence distribution. J Cell Physiol 87:367, 1976 17. Holin DM, Cram LS: An improved microfluorometer for rapid measurement of cell fluorescence. Exp Cell Res 80:105, 1973 18. Kachel V: Methodik und Ergibnesse optischer Formfaktoruntersuchungen bei der Zellvolumenmessungen nach Coulter. Microscop Acta 75:419, 1974 19. Kernell AM, Bolund L, Ringertz NR: Chromatin changes during erythropoiesis. Exp Cell Res 65:1,

ET 23.

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25.

26.

27.

28.

Magath TB, Higgins GM: The blood of the normal duck. Folia Hematol 51:230, 1934 Meinke W, Hall MR, Goldstein DA, Kohne DE, Lerner RA: Physical properties of cytoplasmic membrane-associated DNA. J Mol Biol 78:43, 1973 Modak SP, Chappuis M, Appleby DW: Cytoplaszinc Informational DNA: Fact or Fancy! J Cell Biol 70:140a 1976, (Abstr). Modak SP, Commelin D, Grosset L, Imaizumi MT, Monnat M, Scherrer K: DNA synthesis in circulating erythroblasts of anemic duck: Isolation and properties of nuclear and cytoplasmic nonmitochondrial DNA. Europ J Biochem 60:407, 1975 Phillips DM: Perspective representation of functions of two variables, with overlaid contours. Univ Texas Publication UTJ 5-05-CC086 revised, May 1972 Pollster AW, Ornstein L: The photometric chemical analysis of cells, Analytical Cytology. Edited by RC Mellors. New York, McGraw-Hill, 1959, p 431-518

29.

30.

31.

32. 33.

34.

35.

36.

37.

1971

20. Kreamer PM, Deaven LL, Crissman HA, Van Dilla MA: DNA constancy despite variability in chromosome number. Adv Cell Mol Biol 2:97, 1972 21. Latimer F, Rabinowitch E: Selective scattering of light by pigments in vivo. Arch Biochem Biophys 84:428. 1959 22. Loken MR, Parks DR, Herzenberg LA: Identification of cell asymmetry and orientation by light scattering. J Histochem Cytochem 25:790, 1977

AL.

38.

39.

40.

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