THE

JOURNAL

Copyright

OF

HISTOCHEMISTRY

© 1977 by The

AND

OF

QUANTITATION

Vol. 25, No. 10, pp. 1147-1153,

CYTOCHEMISTRY

Histochemical

Society,

Inc.

1977 in U.S.A.

Printed

CELLULAR

DEOXYRIBONUCLEIC

ACID

BY

FLOW

MICROFLUOROMETRY’ PATRICIA Zoology Department,

B. COULSON,2

Department, Biomedical

Received

ASA

Room 223, Engineering

for publication

ORIN

BISHOP

AND

ROBERTO

LENARDUZZI

Hesler Biology Building (P. B. C.), and Program (A. 0. B., R. L.), University Tennessee 37916

Electrical Engineering of Tennessee, Knoxville,

December

March

30, 1976,

and

in revised

form

22, 1977

This report characterizes for the first time an easy, reproducible means of standardizing the relative fluorescent units normally reported for flow microfluorometry. Absolute values for deoxyribonucleic acid/cell are obtained by using nucleated red blood cells as references. Cells were selected and characterized for the quantitative analysis of deoxyribonucleic acid per cell over a range from 2 pg/cell to 93 pg/cell using literature values for species having nucleated erythrocytes. Fluorescence staining by either acridine-orange (green wavelength) or propidium iodide (red wavelength) gave linear curves over the entire range investigated only when “gain controls” and current are optimized. The range was equivalent to mammalian cell values from 1 N (= 3.5 pg deoxyribonucleic acid/cell) to 28 N (= 91 pg deoxyribonucleic acid/cell). The standard curves obtained with nonmammalian erythrocytes were compared to mammalian free-cell preparations of bovine thymus and liver cells which fell at 6.8 and 6.9 pg deoxyribonucleic acid/cell, respectively. The routine use of these easily obtainable red blood cells will allow ready comparisons on the basis of absolute values for deoxyribonucleic acid per cell for work between experiments, work between staining procedures and dye types and work between laboratories. Many

methods

nucleic are

acid

for

using

available.

as

dye the

analysis

uptake

is and

concentration the timing and the gain settings

high

voltage

to

the

expressed factors

fluorescent

the

the

or

oxyribonucleic acid known deoxyribonucleic to

characterize

obtained propidium are

known

by

to intercalate acid

(2), this

Grant 2

cell, acid/cell

however,

the

fluorescence

double

values

by

may

Portions of this HD06221.

work reprint

FMF

the

de-

stranded

were requests

manner

obtained not

supported should

be

the

relative

for linear.

by HEW be sent.

FMF

deoxyribonucleic

supply values

values

acid,

widest

companies

and

a

which

of deoxyribonucleic possible MATERIALS

standards of were selected

a quantitative

numeric

Author

to whom

with in

the

photomultiplier

the linearity of the fluorescence FMF using acnidine orange (3) or di-iodide (2). These fluorescent dyes

deoxyribonucleic

lished

of incubaand the

laboratories. analyze

pen

biologic

such proce-

tubes. Thus the amount of fluorescence obtained from any single preparation, expressed in relative units cannot be readily compared between runs or between In order to “quantitatively”

to

of dye standard

curve was designed using samples of nucleated erythrocytes (RBCs). Animals were selected which were readily available from commercial

influ-

fixation

evaluate

bound in

intensity

temperature on the machine lazer

To

(FMF)

deoxyribonucleic

invariably units. Many of dye,

dure, tion,

of deoxyribo-

microfluormetry

However,

acid quantitation relative fluorescent ence

the

flow

range AND

had

acid/cell

pubover

(8). METHODS

Erythrocyte samples: Animals were selected (6) that had nucleated red blood cells with a wide range of values for deoxyribonucleic acid/cell (8). These included: Xenopus laevis (African clawed frog = 6.0-6.3 pg deoxyribonucleic acid/cell); Bufo marinus (large South American toad = 11.3 pg deoxyribonucleic acid/cell); Rana catesbeiana (bull frog = 15.2 pg deoxyribonucleic acid/cell); Notophthalamus viridescens (broken, red-lined newt, North American = 93.2 pg deoxyribonucleic acid/cell); Bufo amencanus (American toad = 12.7 pg deoxyribonucleic acid/cell); Rana pipien pipien (Northern leopard frog = 15 pg deoxyribonucleic acid/cell); Rana pipien benlandi (Southern Rio Grande leopard frog = 15 pg deoxyribonucleic acid/cell) and Ambystoma tigninum (salamander = 87 pg deoxyribonucleic acid/ cell) were obtained from Mogul Ed Biological Supply Co., Oshkosh, Wis. Desmognathus fuscus (dusky salamander = 36.0 pg deoxyribonucleic acid/cell) 1147

Downloaded from jhc.sagepub.com by guest on March 18, 2015

1148

COULSON,

BISHOP

and Eurycea bislineata (2-lined salamander = 47.0 pg deoxyribonucleic acid/cell) were both obtained locally (8) on field trips into the Smoky Mountain area of eastern Tennessee by Dr. A. E. Echternacht. Gallus gallus domesticus (leghorn chicken = 2.5 pg deoxyribonucleic acid/cell) was obtained locally. Staining with acridine orange (3): Fresh blood samples were obtained as described below and transfered to isotonic amphibian ringer solution (0.6% sodium chloride) containing 0.76% sodium citrate in order to minimize clotting. Prior to their analysis by FMF, 0.4 ml aliquots (containing approximately 2-5 x 10 erythrocytes) were placed in separate tubes and 1.0 ml of a plasma membrane disrupting solution was added (0.1% (v/v) Triton X100 (Sigma Chemical Co., St. Louis, Mo.), 0.2 M sucrose, 0.2 mM EDTA, and 2 x 10_2 M citrate phosphate buffer; pH 3.0). The cells were incubated 1 mm (25#{176}C) and then 2 ml of the dye added (0.002% acridine orange, (Sigma #A-2886), 0.1 M NaC1, and 102 M citrate-phosphate buffer, pH 3.8). After 10 mm of equilibration with the dye (25#{176}C), the samples were cooled in an ice bath or measured immediately by FMF. Acridine-orange stained cells were stable (0-3#{176}C) for up to 4 hr. Inspection of the cells under phase and fluorescent microscopy revealed that the hypotonic dye or Triton X-100 did not lyse the cells and that the green fluorescence was localized exclusively in the cell nucleus. The red fluorescence is located primarily in the nucleoli and in the cytoplasm. Treatment with EDTA allowed differential staining of deoxyribonucleic acid by denaturing any double stranded nibonucleic acid (ribosomes, nuclear components, etc.) that would compete for acnidine orange binding and increase background fluorescence (3, 7). Fixation followed by staining with propidium diiodide: Small blood samples were taken directly from small incisions on the top of the back foot of the amphibians. This allowed the same animal to be used over a 6-month period for multiple samples. Animals were maintained at 25#{176}C, in plastic containers containing a 2 inch layer of water in the bottom. They were fed, 1-2 times a week, a variety of fresh insects or by force feeding them bovine liver, etc. Amphibians frequently live to be 15-25 years old and are fairly disease free (6). Nucleated blood was transferred from the incision with a disposable pipette by flushing with amphibian saline +Na citrate (0.2 ml) and placed directly into a fixative (4 ml, 70% ethanol). Samples were vortexed and fixed at 3#{176}C for 30 mm (3). After centrifugation (5 mm, 700 x G, 3#{176}C), the fixative was aspirated off, the cell pellet vortexed and the staining solution added (0.05 mg/ml propidium diiodide; Calbiochem #537059, mol. wt. = 668.4) in 0.1% hypotonic sodium citrate (2). Stained cells were stable in this solution (3#{176}C, in the dark) for 1-

AND

LENARDUZZI

24 hr. This staining procedure has been utilized for many different cell types including CHO, L929, ascites L1210 cells (2). Fluorescent analysis: Cells were measured in a flow microfluorometer (Bio/Physics Cytofluorograf, Mahopac, N.Y., Model 4801) which had been interfaced with a Texas Instruments (Model 980A) computer (5). Fluorescence signals were generated by cells in the instrument as they flow in a fine column and cross a 488 nm argon-ion laser. The red (F 600 nm) and the green (F 530 nm) fluorescence wavelengths from each cell are separated optically and quantitated by separate photomultipliers. This FMF-Computer system is capable of storing four parameters (cell number, cell size, fluorescence in green and fluorescence in red wavelengths) for each individual cell up to a maximum of 5000 cells. Following sample analysis the data is sorted into 1024 channels and expressed as amplitude/distribution histograms or the absolute number of cells in each channel. Mode of display is optical (oscilloscope, CRT terminal or printed copy). The histograms can display cell number compared to any one of the other 3 parameters (Table I-Ill). RESULTS

ties

AND

Treatment of cells of deoxyribonucleic

fluorescent

dye,

DISCUSSION

containing known acid/nucleus

acridine

orange,

quantiwith the

was

investi-

gated in order to evaluate the linearity uptake and fluorescence over the wide deoxyribonucleic acid values measurable Cytofluorograf. cent values after

Figure several

for

staining

with

(25#{176}C)and

1-50 pg/cell correlation gain cell curve. on the decreasing Several arations

acnidine-orange at

for

a high

were

expressed per point of deoxyribonucleic

gain

(x) and a this high

values of deoxyribonucleic range of 50-100 appeared but

These

not

the

higher

linear the

“linear” values

(see “materials in Figure 2.

stain procedure procedures using

was chosen non-fixed

to allow more and analysis.

be

nucleated erythrocyte with the fluorescent

pidium di-iodide are illustrated

time between Relative red

Downloaded from jhc.sagepub.com by guest on March 18, 2015

on

acid/ the

of

the

portion could

portion of the curve gain to 60/100 (data not

different treated

10 mm setting

as ranges with (regression analacid/cell of

gave a slope of y = 1.64 coefficient, R = 0.93). At

setting, in the

“plateau”

1 illustrates the fluoresdifferent RBC standards

analyzing

(80/100). Values 2-3 determinations ysis for values

of dye range of in a

and The

included only by shown). dye,

preppro-

methods”), fixation

(2) over published cells (1, 4) in order sample preparation fluorescence inten-

+

QUANTITATION

OF

CELLULAR

DNA

BY

TABLE (A) Fluorescent 100.00%

Channel

= Number

OF SAMPLES

ofCells

and

TAKEN.

[I]

FLOW

1149

MICROFLUOROMETRY

I (B)

Amplitude

CHICKEN

RBC:

Distribution (A) CELLS

Histograms#{176}

PER CHANNEL

5000 OF 5000 CELLS 0000#{176}0029 0006#{176}0731 0O12#{176}0056 0018#{176}0O17 0024#{176}0012 0030=0013 0036#{176}0O12 0042=0014 0048=0010 0054=0002 0060#{176}0000 0066#{176}0000 0072#{176}0000 0078=0000 0084=0001 0090=0000 0096=0000 0102#{176}0001 0108=0004 0114=0000 0120=0001 0126=0000

0001#{176}009O 0007#{176}0598 0O13#{176}0052 0O19#{176}O018 0025#{176}OO19 0031#{176}0017 0037#{176}0014 0043#{176}0018 0049=0007 0055=0003 0061 #{176}0002 0067=0000 0073=0001 0079=0000 0085=0000 0091 =0000 0097#{176}0000 0103=0001 0109=0001 0115#{176}0000 0121=0000 0127#{176}0000

0002#{176}0271 0008#{176}0424 0014#{176}0O29 0020#{176}0O14 0026#{176}0015 0032#{176}0013 0O38#{176}0013 0044=0007 0050#{176}0008 0056=0004 0062#{176}0002 0068=0001 0074=0000 0080=0000 0086=0000 0092=0000 0098=0000 0104=0003 0110=0000 0116#{176}0000 0122=0000 0128=0000

0003#{176}0366 0009=0242 0015#{176}0O34 0021#{176}0022 0027#{176}0008 0033#{176}0013 0039#{176}0017 0045=0007 0051#{176}0003 0057=0003 0063#{176}0000 0069#{176}0003 0075=0003 0081 =0001 0087=0000 0093=0000 0099=0000 0105=0002 0111=0000 0117=0000 0123=0000

I] 0720 0690 0660 0630 0600

CHICKEN

0004#{176}0541 OO1O#{176}0157 0016#{176}0024 0022#{176}0020 0028#{176}0014 0034#{176}OO11 0040#{176}0013 O046#{176}OO1O 0052=0002 0058#{176}0000 0064#{176}0001 0070#{176}0000 0076=0000 0082=0000 0088=0000 0094=0001 0100=0000 0106=0001 0112=0000 0118=0000 0124=0000

RBC:

(B)

0005#{176}0736 OO11#{176}0098 0017#{176}0022 0O23#{176}0015 0O29#{176}0013 O035#{176}OO11 0041=0014 0047#{176}OO1O 0053#{176}0005 0059#{176}0004 0065=0000 0071 #{176}0000 0077=0001 0083=0000 0089=0000 0095=0000 0101=0001 0107=0004 0113=0000 0119#{176}0000 0125=0000

HISTOGRAM

+ + ++ ++ ++

0570 0540 0510 0480

+++ ++++ ++++ ++++

0450 0420 0390 0360 0330 0300 0270 0240

+++++ +++++ ++++++ ++++++ ++++++ +++++++ ++++++++

0210

++++++++

0180 0150 0120 0090 0060 0030 0000

++++++++ +++++++++ +++++++++ +++++++++++ +++++++++++ +++++++++++++ +

+

0000 0000

+

+++++++++++++++++++++++++++++++++++++++++++++++++++++++1-1+++++++++++++++++ +

0010

+

0020

+

0030

+

0040

+

0050

+

0060

0070

++++++++++++++++++++++++++++++++++++++++++++++++++++++ +

0080

+

+

0090

+

0100

+

0110

0120

o In Tables I-Ill the erythrocytes stained with acridine orange are illustrated after analysis by FMF. Crude data are shown as they appear on the CRT terminal and on the computer printout. (A) Shows cell numbers (left) per channel (right). (B) Shows histograms comparing any 2 parameters. Shown here are cell numbers on Y (vertical) axis, while the X (horizontal) axis gives relative fluorescent intensity in the green wavelength expressed on a scale of 1024 total channels which are grouped with 8 channels per point for 127 divisions. [I] Chicken RBCs, stained with acnidine-orange and analyzed for green fluorescence: (A) Green fluorescent channel = number of cells in that channel. (B) Cell numbers (Y axis) are graphed in steps of 30 and green fluorescence (X axis) shows a peak DNA/cell in channels 5-6.

sity

was

known

RBC values of deoxyribonucleic Values were expressed as ranges

cell.

determinations

plotted

on a total

per

point

scale

of 1-127

(regression

against

of the

points

acid/ with 2-3

correlation gain setting

analysis

range

1/100

gave

a slope

pg/cell

Downloaded from jhc.sagepub.com by guest on March 18, 2015

of y = 1.03

(X)

and

a

R = 0.987). By using a all RBC values in the fell on a single slope.

coefficient of 60/100,

of

1150

COULSON,

BISHOP TABLE

(A) Fluorescent

= Number

Channel

100.00% OF SAMPLES TAKEN. 5000 of 5000 0000=0053 0001 =0000 0006=0001 0007=0000 0012=0011 0013=0017 0018=0183 0019=0306 0024=0313 0025=0235 0030=0115 0031=0094 0036=0065 0037=0053 0042#{176}0044 0043=0029 0048=0018 0049=0022 0054=0015 0055=0016 0060=0010 0061=0011 0066=0009 0067=0003 0072=0000 0073=0003 0078=0003 0079=0003 0084=0000 0085=0002 0090=0002 0091 =0002 0096=0000 0097=0003 0102=0002 0103=0003 0108=0004 0109=0003 0114=0018 0115=0007 0120=0002 0121=0001 0126=0000 0127=0003

LENARDUZZI

AND

of Cells

II

and

(B) Amplitude

II. 0002=0002 0008=0002 0014=0027 0020=0409 0026=0210 0032=0073 0038=0057 0044=0032 0050=0012 0056=0005 0062=0008 0068=0006 0074=0002 0080=0003 0086=0001 0092=0000 0098=0001 0104=0000 0110=0004 0116=0008 0122=0001 0128=0000

R. PIPIEN

RBC:

0003=0001 0009=0003 0015=0043 0021#{176}0436 0027=0161 0033=0066 0039=0044 0045=0031 0051=0017 0057=0007 0063=0004 0069=0006 0075=0005 0081=0003 0087=0005 0093=0001 0099=0000 0105=0003 0111=0006 0117=0006 0123=0001

II.

Distribution (A) CELLS

0004=0000 0010=0002 0016=0067 0022=0423 0028=0140 0034=0073 0040=0030 0046=0023 0052=0011 0058=0011 0064=0004 0070=0003 0076=0005 0082#{176}0002 0088=0003 0094=0000 0100=0000 0106=0002 0112=0007 0118=0004 0124=0001

R. PIPIEN

RBC:

of Histograms#{176} PER

CHANNEL

0005=0000 0011 #{176}001 2 0017=0118 0023=0366 0029=0127 0035=0061 0041#{176}0032 0047=0017 0053=0014 0059=9997 0065=0004 0071=0006 0077=0003 0083=0004 0089=0001 0095=0003 0101=0003 0107=0003 0113=0007 0119=0005 0125=0000

(B) HISTOGRAM

++

0420 0400

0380 0360 0340 0320 0300 0280 0260 0240 0220 0200 0180 0160 0140 0120 0100

++++++++

0080

0060 0040 0020 0000

+ + +++ +

+

+

+

+

+

+

+

0000

0010

0020

0030

0040

0050

0060

0070

0000 +

+

0080

+

0090

+

0100

+

0110

0120

o [II] Rana pipien pipien RBCs, stained with acridine-orange: of cells in that channel. (B) Cell numbers are graphed fluorescence (X axis) in channels 21 and 22.

Using pipien gave

acid/cell, leopard

propidium values

pipien

frog)

gave

values cell.

acid/cell values The

Mogul of These

di-iodide northern pg

14.3-14.8

of

R.

bonucleic frog) gave acid/cell. tans;

(small

pipien

2) R. frog)

(Fig. leopard

deoxyribonucleic

guished thesis using

riogrande (large southern values of 14-15.0 pg deoxyri-

cell.

and R. catesbeiana of 14.8 pg deoxyribonucleic

dyes orange

green

Ed)

stain

was

13.0-14.0 preparations

grass an pg

(bull

frog (Rana clamiunknown and gave

deoxyribonucleic could

not

acid/ be

distin-

(A) Green fluorescent steps of 20 and show

in

statistically in Figure

2 and

deoxyribonucleic

Analysis

showed gave

channels = number a maximum green

as indicated by the parenwere therefore, graphed acid/cell

values

of 15 pg/

the raw data obtained with the the cells stained with acridine a wider range or spread of fluores-

of

cence and a lower correlation did the samples stained with

coefficient propidium

The procedure for individual handling

orange samples

Downloaded from jhc.sagepub.com by guest on March 18, 2015

acridine of the

2

than iodide. required prior

to

QUANTITATION

OF

CELLULAR

DNA

BY

TABLE (A) Fluorescent

Channel

= Number

100% OF SAMPLES TAKEN. 5000 OF 5000 CELLS 0001=0000 0000=0001 0006=0005 0007=0001 0012=0001 0013=0001 0018=0001 0019=0001 0024=0001 0025=0001 0030=0000 0031=0000 0036=0000 0037=0000 0042=0001 0043=0001 0048=0001 0049=0001 0054#{176}0007 0055=0005 0060=0013 0061=0009 0066=0010 0067=0007 0072=0010 0073=0018 0078=0012 0079=0014 0084=0012 0085=0016 0090=0013 0091=0021 0096=0012 0097=0016 0102=0027 0103=0025 0108=0034 0109=0068 0114=0424 0115=0675 0120=0099 0121=0048 0126=0001 0127=0005

of Cells

FLOW

MICROFLUOROMETRY

(B) Amplitude

Distribution

III and [III]

NEWT

RBC:

(A) CELLS

Histogramso

PER

CHANNEL -

0002=0001 0008=0001 0014=0003 0020=0000 0026=0000 0032=0000 0038=0000 0044=0001 0050=0003 0056=0009 0062=0007 0068=0020 0074=0007 0080=0017 0086=0015 0092=0011 0098=0020 0104=0025 0110=0061 0116=0836 0122=0013

0003=0001 0009#{176}0002 0015#{176}0002 0021=0001 0027=0000 0033=0002 0039=0000 0045=0001 0051=0001 0057#{176}0003 0063=0011 0069=0011 0075=0020 0081=0012 0087=0010 0093=0015 0099=0015 0105=0027 0111=0075 0117=0759 0123=0008

[III] 0000

1151

NEWT

0004=0002 0010=0006 0016=0001 0022=0001 0028=0000 0034=0001 0049=0000 0046=0002 0052=0001 0058=0016 0064=0011 0070=0015 0076=0011 0082=0011 0088=0022 0094=0009 0100=0018 0106=0025 0112=0137 0118=0471 0124=0001

RBC:

0005=0000 0011=0003 0017=0003 0023=0000 0029=9991 0035=0001 0041=0001 0047=0002 0053=0004 0059=0007 0065=0012 0071=0013 0077=0009 0083=0016 0089=0010 0095=0020 0101=0024 0107#{176}0045 0113=0211 0119=0213 0125=0000

(B) HISTOGRAM

++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ +

+

0000

+

0010

+

0020

+

0030

+

0040

0800 0750

+

0700

++

+

0050

+

0060

0070

++

0650 0600 0550 0500 0450 0400 0350 0300 0250 0200 0150 0100 0050 0000 +

+

0080

o [III] channel maximum

and

a greater

(i.e.,

which

probably

caused

photomultiplier

settings gives

high

increases an increase

fluorescent acid/cell

the in

voltage

slope of the the fluores-

values in standards

the

higher were not

linear dashed

portion of this line (See Fig.

to 60/100 the entire

(not shown) range (Fig.

gave 1).

the

of deoxyribonucleic

In summary, acid/cell

no fixative the

gain

cence). The deoxyribonucleic

gain over

+

0110

variation.

Increasing

on the by the

+

0100

0120

Newt (Notophthalamus viridescens) RBCs stained = number of cells in that channel. (B) Cell numbers green fluorescence (X axis) in channel 116.

analysis

or the values

+

0090

by

analysis

fluorescent

curve as indicated 1). Decreasing the

staining

linear

with

values

either

with acridine-orange: (Y axis) are graphed

acridine

orange

or

(A) Green fluorescent in steps of 50 and show

propidium

di-iodide

can

a

be

easily quantitated using erythrocyte standards which cover a wide range of deoxyribonucleic acid/cell values. were used to

When establish

nucleated red blood the appropriate

and range for the standard curve, sis could give a linear fluorescence bonucleic

acid

from

acid/cell. that not

However, all settings

bonucleic perative

acid that

1-93

Downloaded from jhc.sagepub.com by guest on March 18, 2015

FMF analyfor deoxyri-

deoxyribonucleic

it is important to point out will give a linear deoxyri-

to fluorescence each setting

deoxyribonucleic

pg

cells slope

acid

be

standards

graph. examined such

It

is imusing as

nu-

1152

COULSON,

BISHOP

AND

LENARDUZZI

0 4

C 0 -J

z Lu Lu

0

20

30

40

50

60

70

80

90

100

DNA/CELL (pg) 1. Acridine-orange stained deoxyribonucleic acid per cell. Nucleated erythrocytes were stained with 0.002% acridine-orange in citrate-phosphate buffer and analyzed by FMF for their relative green fluorescence (A 530 nm). Values are expressed as ranges of 2-3 points and the line drawn by regression analysis using a Hewlett Packard programable computer (slope y = 1.64 (X), R = 0.93). FMF was performed at gain settings of 80/100 and constant current = 5.5 volts. Standard curve has been repeated four times. Because there were no points between E. Bislineata and newt in this particular experiment it is uncertain where the break in linearity should begin, therefore this region was connected by a dashed line. However, the break in linearity is not due to limited dye availability since reduction of the gain settings to 60/100 decreases the slope and increases the linear range to 1-93 pg/cell (not shown). DNA, deoxyribonucleic acid; A. 0., acridmne-orange. FIG.

a:

0 -J

0 Lu

DNA/CELL

(pg)

di-iodide stained deoxyribonucleic acid per cell. Nucleated erythrocytes were stained with propidium di-iodide and analysed by FMF for their relative red fluorescence (A 600 nm). Values are expressed as ranges of 2-3 points per species and the line drawn by regression analysis using a Hewlett Packard programable computer (slope y = 1.03 (X), correlation coefficient = 0.987). Mammalian thymus (bovine) is indicated at 7.0 pg deoxyribonucleic acid/cell. FMF was performed at gain settings of 60/100 and constant current = 5.5 volts. Standard curve repeated five times. DNA, deoxyribonucleic acid; P.1., propidium di-iodide. FIG.

2.

Propidium

Downloaded from jhc.sagepub.com by guest on March 18, 2015

QUANTITATION cleated RBCs experimental malian

cells

using

either

iodide cells

6.8

CELLULAR

before these settings samples. Comparison to these

nucleated

acridine

orange

stain, at

OF

placed pg

the

are of

RBC

were 14-15

2N

di(bovine)

acid/cell

5.9-6.8 pg/cell, respectively from Rana clamitans (green the

standards,

thymus

deoxyribonucleic

demonstrated for pg deoxyribonucleic

used on mam-

or propidium

2) and 2N liver (bovine) cells (Fig. deoxyribonucleic acid/cell which agreement with literature values and liver (bovine) deoxyribonucleic 7.1 and rocytes

DNA

first time acid/cell.

(Fig.

1) at 6.1 pg is in good for thymus acid of 6.4(5). Erythgrass frog) to contain

ACKNOWLEDGMENTS

The

authors

tion for Hansen of samples,

wish

the assistance and James to

Dr.

to express

their

apprecia-

of James Gillespie, Dale Ireland in the preparation A.

E.

Echternacht

for

the

salamander samples, to Dr. Aza Bishop for assistance interfacing and for use of the Texas Instrument Computer plus CRT system, and to Nancy Crews for preparation of the manuscript.

BY

FLOW

MICROFLUOROMETRY LITERATURE

1153

CITED

1. Cram LS, Gomez ER, Thoen CO, Forslund JC, Jett JH: Flow microfluorometric quantitation of the blastogenic response of lymphocytes. J Histochem Cytochem 24:383, 1976 2. Crissman HA, Oka MS, Steinkamp JA: Rapid staining methods for analysis of deoxyribonucleic acid and protein in mammalian cells. J Histochem Cytochem 24:64, 1976 3. Darzynkiewicz Z, Traganos F, Sharpless T, Melamed MR: Lymphocyte stimulation: a rapid multiparameter analysis. Proc Natl Acad Sci USA 73:2881, 1976 4. Krishan A: Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol 66:188, 1975 5. Lenarduzzi R, Biship A0: MicrofluorometerComputer System, Abstract, Amer Conf Eng in Medicine and Biology 28th, 1975 6. Nace G: Amphibians, Guidelines for breeding, care and management of laboratory animals. National Academy of Science, Washington, DC, 1974, p 153 7. Ornstein L, Ansley HR: Spectral matching of classical cytochemistry to automated cytology. J Histochem Cytochem 22:435, 1974 8. Sober HA: Deoxyribonucleic acid content per cell of various organisms, Handbook of Biochemistry and Molecular Biology, CRC, Cleveland, Ohio, 3rd Ed, 1976, p 284-306 9. Snyder DH: Amphibians and Reptiles of Land Between the Lakes. Tennessee Valley Authority Publication, 1972

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