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Volume and Osmotic Properties of Human Neutrophils
By H. Ping Ting-Beall, David Needham, and Robert M. Hochmuth
Quantitative models describing the dynamics of human
neutrophils in the microcirculation require accurate morphometric parameters such as volume and surface membrane area. Using both a micropipette technique and video
light microscopy (LM) to measure the diameters of the
spherical cells, w e have accurately determined the volume
of the human neutrophil. Our value, 299 & 32 gm3, is in
good agreement with our earlier results, but 55% larger
than that reported by Schmid-Schonbein et a1 (Blood 56:
866, 1980). However, the measurements of SchmidSchanbein et a1 were based on the actual mass of the cells
derived from transmission electron microscopic (TEM) images. The membrane surface area, at lysis, was calculated
to be 2.6 times its initial projected area. After lysis, the
cells do not reducetheir size, indicative of the possibility of
a F-actinnetworkformationthat would stiffenthe structure.
Further, we show that neutrophils behave as ideal osmometers when exposed to anisotonic solutions at 21 “C, as
predicted by the Boyle-Van’t Hoff relationship. The calculated Ponder’s value, R, is 0.77, which correspondsto 77%
of the cell volume being osmotically active under isotonic
conditions. However, at 37”C, the cells are able to regulate
their volumes toward the original volumes after an osmotic
stress.
0 1993 by The American Society of Hematology.
T
bin). In contrast to the RBC,77% ofthe volume of neutrophils
is osmotically active water. We also show that the ability of
cells to regulate their volume after osmotic stress is temperature-dependent.
HE HUMAN NEUTROPHIL has recently come under
scrutiny with regard to its rheologic, mechanical, and
geometric properties. We, along with other researchers, are
using micropipette manipulation techniques to deform the
cell. The total aspiration of a single cell into a micropipette
allows a more accurate measure of cell volume than a simple
measurement of the diameter of the spherical cell. Using the
micropipette technique, we found that the average isotonic
cell volume of 150 neutrophils from five individuals is 280
k 30 pm3.’ This value is 47% larger than that obtained by
Schmid-Schonbein et a1 via a transmission electron microscope (TEM)-stereologytechnique? We therefore performed
a more careful series of measurements in which we used micropipettes to measure the volumes of cells held in a static
position. The cells were in equilibrium with the suspending
media that were isotonic, hypotonic, and hypertonic with
respect to blood plasma. We found that the measured cell
volumes were consistent with our earlier measurements, and
the membrane surface area, at lysis, in a slow swelling cell
was calculated to be 2.6 times its initial projected area. After
lysis, the cell does not reduce its size, which is indicative of
a possible F-actin network formation that would stiffen the
structure.
Equilibrium cell volume measurements induced by solution osmolarity changes yield estimates of the percentage of
osmotically active water. The remaining volume of material
in the cell is i n a ~ t i v e For
. ~ the red blood cell (RBC),the
osmotically active fraction of the isotonic volume is approximately 0.57; indicating that 43% of the cell volume is occupied by impermeable salts and protein (largely hemogloFrom the Department of Mechanical Engineering and Materials
Science, Duke University, Durham, NC.
Submitted July 13. 1992; accepted January 4, 1993.
Supported by National Institutes of Health, National Heart, Lung,
and Blood Institute Grant No. HL23728.
Address reprint requests to H . Ping Ting-Beall, PhD, Department
of Mechanical Engineering and Materials Science, Box 90300, Duke
School of Engineering, Durham, NC 27708-0300.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 I993 by The American Society of Hematology.
0006-49 71/93/8110-0033$3.00/0
2774
MATERIALS AND METHODS
Cell Preparation
Human neutrophils were isolated from the peripheral blood of
three healthy volunteers in a sterile environment as described previously.sApproval was obtained from the Institutional Review Board
for these studies. Volunteers were informed that blood samples were
obtained for research purposes, and that their privacy would be protected. Whole blood drawn into an EDTA (K,) vacutainer (Becton
Dickinson, Rutherford, NJ), was centrifuged at 300g for 25 minutes
at 25°C to remove the majority of erythrocytes. The plasma and
buffy coat were diluted to 50% with modified endotoxin-free Hanks
balanced salt solution (HBSS; Sigma, St Louis, M O no Caz+ and
MgZ+)and carefully layered over Ficoll-Hypaque gradients (Sigma
Histopaque-1077 and -1 1 19) having densities of 1.077 and 1.1 19,
respectively, at 25°C. After a 20-minute centrifugation at 800g and
2 5 T , the neutrophils at the 1077/1119 interface were collected, and
washed twice with 10X volume HBSS, and finally resuspended in
50%autologous plasma/HBSS to prevent adhesion to glass surfaces.
The cells were 99% viable as determined by trypan blue exclusion
test, and 90% passive as determined visually by the spherical shape
and absence of pseudopods.
The osmotic pressures of all the solutions were measured using a
freezing-point depression osmometer (Advanced Instruments, Inc,
Needham Heights, MA). Increase and decrease of plasma solution
osmolality was done by addition of NaCl and distilled water, respectively, to the isotonic solution (300 mOsm).
Volume Measurement of Spherical Cells by Light
Microscopy
The cells were placed in a microchamber on the microscope stage
and examined with a Leitz Diavert bright-field microscope (Rockleigh,
NJ) equipped with two 1OX eyepieces and a vertical 25X eyepiece
on a trinocular head and a 40X, 0.65-numerical apperature (NA)
long working distance Nikon objective (Garden City, NY). For comparison, cell images were also checked with a lOOX oil immersion
objective that had a NA of 1.25. Illumination was provided by a 200W mercury lamp with a 4,358-nm monochromatic filter. Experiments
were recorded on videotape with a video camera (Dage-MTI, Michigan City, IN) mounted above the vertical eyepiece. Geometric measurements were performed using a position analyzer (Vista Electronics,
La Mesa, CA) via a contour synthesizer (Model IV-53; For-A, West
Newton, MA). The diameter of the cell was the average value of the
Blood. Vol81, No 10 (May 15). 1993: pp 2774-2780
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2775
NEUTROPHIL VOLUME AND OSMOTIC PROPERTIES
I
I
I
pulled to a fine point and broken by quick fracture to the desired tip
dimension. Only the pipettes with constant diameters within 25 pm
of the tip were used. The pipette diameter was measured optically
by inserting an ink-tilled pipette into the immersion oil (Fig 2b). and
by scanning electron microscopy (Fig Zc) of the same pipette. Both
techniques gave the same pipette diameter. The pipette was filled
with NaCl solution that was equiosmotic with the equilibnum cell
bathing solution (le. 150. 300. or 450 mOsm), and was connected to
a manometer via water-filled tubing. Cells were aspirated into the
pipette into a sausage shape as shown in Fig 1. either by mouth
suction or by a simple displacement of a synnge. Expenments were
video-recorded for subsequent volume analyses. For temperaturedependent studies of volume regulation. temperature-controlled
double chambers were used. Cells in a 300-mOsm solution chamber
were transferred with a micropipette to the chamber containing a
100-mOsm solution.
c
C
1
I
I
I
I
I
I
I
i
I
I
I
I
I* - - - - -
L - - - - - e
I
Fig 1. Geometry of a neutrophil outside and inside of a pipette.
Video micrographs of a cell (a) at the bottom of the chamber and
(b) completely inside of a pipette. (c) Shows the geometry of a cell
inside of a pipette and depicts a cylinder enclosed by two hemispherical caps.
horizontal and vertical dimensions of the cell. Sharp focus of the
outer edge of the cell shows a thick 0.4-pm dark ring (Figs I and 2).
The contour synthesizerenhances the image, and this thick diffraction
ring is reduced to a sharp line that falls between the inner and outer
edges of the ring. Because of the small size (0.2 pm) and uncertainty
as to where to place the calipers to give the absolute measurement
ofcell diameter, we calibrated our video caliper system by measuring
a sample of polystyrene latex beads of known diameter (9.6 ? 0.08
pm: Polyscience. Wamngton. PA). Our measurement of the beads
gave a value of 9.5 ? 0.4 for the diameter, which is 170smaller than
that provided by the manufacturer, who used TEM to measure the
bead size. To check further the relationship between the geometric
border and the diffraction pattern of a cell. we measured the epifluorescent and phase-contrast images of individual RBCs and RBC
ghosts stained with a lipophilic carbocyanine membrane probe, DilC l8:3 ( l. I‘dioctadecyl-3.3.3’.3’-tetramethyl-indocar~yanine perchlorate: Molecular Probes, Eugene, OR). The two white lines in Fig
la represent the geometric border of all the cells we measured.
A d i c r o p i p i ~ Munipirlution
i~
Micropipettes were formed from I-mm outer diameter and 0.75mm inner diameter glass capillary tubing (A-M System. Everett, MA)
Fig 2. Geometry and dimension of a pipette as shown (a) optically
in 50% plasma, (b) optically in immersion oil after being filled with
ink, and (c) by scanning electron microscopy after gold-palladium
spluttering.
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2776
TING-BEALL, NEEDHAM, AND HOCHMUTH
The geometry of a neutrophil in the pipette is illustrated in Fig IC.
The geometry depicts a cylinder capped by two hemispherical segments. Thus, the total volume of the cell can be calculated from the
equation V = nR*(L- 2R) + (4/3)*R3.
Scanning Electron Microscopy
Cells were prepared by first depositing them on 12-mm glass disks
previously treated with Cell-Tak (Collaborative Biomedical Research,
Bedford, MA). After I minute, the cells were fixed with 0.1%glutaraldehyde for 1 hour at room temperature followed by another hour
in 1% glutaraldehyde. After washing the cells with buffer, the cells
were postfixed in 1% Os04 for I hour in veronal acetate buffer at pH
7.4. Dehydration of the cells was performed in a graded series of
acetone and then critical-point-dried using liquid CO, in a Ladd
critical-pointdryer (Burlington, VT). Disks with monolayers of cells
were mounted on aluminum stubs using a conductive paint, and
coated with a thin layer (-20 nm) of gold-palladiumusing a Hummer
V sputterer (Anatech, Ltd, Alexander, VA). Samples were stored under
vacuum until they were examined and imaged in a Philips 501 scanning electron microscope (Mahwah, NJ) at 15 kV.
RESULTS
Cell Response in Anisotonic Solutions
Spherical cell measurements by light and electron microscopy. Table 1 shows the average volume of human neutrophils of our studies, as well as those reported earlier by other
laboratories. The average cell volumes from our study at 300
mOsm were 294 +- 36 pm3 and 304 27 pm3, which is much
larger than the value reported by Schmid-Schonbein et al,2
who used both light microscopy (LM) and TEM-stereology
to obtain a value of 190 pm3. However, our value is in good
agreement with our earlier report,’ as well as that of Evans
and Yeung.6 The average cell diameter obtained by Evans
and Yeung via LM (sphere) was 8.5 pm, which corresponds
to a volume of 322 pm3 at 290 mOsm. To assure that removal
of plasma during isolation procedure does not perturb the
cells, measurements on cell volume were performed on neutrophils collected from the buffy coat before isolation procedure. The cell dimensions of those cells were identical to
those that were isolated by Ficoll-Hypaque gradient.
Video micrographs of a group of neutrophils are shown
suspended in 70-, 150-, 300-, and 450-mOsm solutions (Fig
3a through d). For comparison, we have also performed the
morphologic analyses of neutrophils from scanning electron
microscopy (SEM) at equivalent osmolalities (Fig 3e through
h). The average cell volume at 300 mOsm from SEM was
167 ? 29 pm3, approximately 43% smaller than the number
obtained with LM. At all osmolalities, the cells that were
fixed, dehydrated, and critical-point-dried were generally
smaller. It has been shown that the greatest cell shrinkage
actually occurs during critical-point
The cells fixed
in low osmolality (Fig 3e) did not exhibit the same type of
microvilli or foldings as the ones fixed in higher osmolalities
(Fig 3f through h). However, the surfaces were not perfectly
smooth; they were either undulating or had small foldings.
Cell volume by micropipette measurement. Because of
defined geometry (Fig I c), the total aspiration of a single cell
into a micropipette allows a more accurate measurement of
cell volume than simply measuring the diameter of the
spherical cell. Typically, a k0.2-pm error in cell radius would
*
Table 1. Neutrophil Volume Data From Various Sources
Source
Schmid-SchMbeinet al*
Bagge and Brhemark’4
Evans and Yeung’
Needham and
Hochmuth’
Present study
Osmolality
(mOsm)
Volume
(rm3)
Technique
310
290
290
300
190
382
322
281 t 3 0
LM (sphere) or
TEM-stereology
LM (sphere. in vivo)
LM (sphere)
LM (micropipette)
300
294 ? 36
304 i 27
LM (sphere)
LM (micropipette)
300
Abbreviations LM, light microscope. with the cell either in free solution and shaped
like a sphere or in a micropipette and shaped like a sausage, TEM, transmission
electron microscope
lead to a 15% error in cell volume, whereas the same error
in the length of the cell in the micropipette gives an error of
1%. Thus, we have also used this technique to measure cell
volumes in isotonic and anisotonic solutions. The results of
these studies are shown in Fig 4. In this figure, we also plotted
the data from spherical cells using LM and SEM. It is interesting to note that the volume measurements from undeformed spherical cells and micropipette-deformed cells are
close. Thus, careful studies by measuring the size of the
spherical cells with appropriate calibration of the optical systems can give accurate volume measurements. In comparison,
SEM results show that the cell volumes were smaller at all
osmolalities.
If we assume that the number of osmotically active particles
(mainly inorganic ions) within a neutrophil remains constant
as the cell swells and that only a fraction, R, of the isotonic
volume is osmotically active, then the relationship between
the volume, V, of the cell relative to the isotonic volume and
the osmolality, P, of the suspending medium relative to the
isotonic osmolality (300 mOsm) can be derived? V = (R/P)
(1 - R). Then the plot of V versus I/P will be a straight
line if the cell behaves as an “ideal” osmometer. Figure 5
shows that at 2 1 “C the neutrophil volume varies linearly with
the inverse of the osmolality over a wide range. The slope,
R, is also referred to as Ponder’s value and is 0.77 in this
case. Thus, we find that 77% of cell volume under isotonic
condition is osmotically active. This value is consistent with
the published values that range from 0.8 and 0.9 for human
lymphocytes* to 0.6 for granulocyte^.^
Cell surface area estimation. At 60 mOsm, the cells
swelled and lysed within 30 minutes. The volume just before
lysis was estimated to be approximately 1,200 pm3 and the
final area was calculated to be 2.6 times the initial area (Table
2). However, when the cells were suspended in distilled water
(0 osmolality) they lysed in 2 minutes and the projected surface area at lysis was only 2.1 times the initial area. This
number is consistent with the value reported by Evans and
Yeung,6 who used the micropipette to estimate the surface
area. After lysis in distilled water, the membrane appeared
to be resealed and returned almost to its spherical shape with
a much smaller diameter (Fig 6). The cell, lysed in a 60mOsm solution, did not reduce in size after lysis. Figure 7
shows that most of the granules appeared to adhere to the
plasma membranes.
+
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NEUTROPHIL VOLUME AND OSMOTIC PROPERTIES
2777
Fig 3. Video micrographs of passive human neutrophils at four different osmolalities: (a) 70 mOsm, (b) 150 mOsm, (c) 300 mOsm, and
(d)450 mOsm. At these resolutions, the membranefoldings are not visible. Scanning electron micrographsof cells suspended in equivalent
osmolalities are shown in e through h. On the whole, the cells were much smaller in size, and at very low osmolality (e), the membrane
had lost its foldings. although they were not perfectly smooth.
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2778
TING-BEALL, NEEDHAM, AND HOCHMUTH
1400
ell
sis
1200
1000
-E :
>" 5
*0°
600
400
200
0
0
100
200
300
400
500
Osmolality
(mOsm)
0
Fig 4. Average neutrophil volume versus the osmolality of the
suspending media. ( 0 )Data taken from pipette measurement, (0)
data calculated from the diameter measurement of spherical cells,
and (B) data from scanning electron microscopy.
Volume Regulation in Neutrophils
Regulatory volume decrease (RVD) following application
of hypoosmotic condition has been described in many cell
types including
Neutrophils also showed a
volume adjustment after osmotic shock. The typical result
shown in Fig 8 is for a neutrophil transferred from an isotonic
solution to a chamber containing a 100-mOsm solution at
37°C. The cell swelled rapidly (within I minute) and then
slowly decreased in volume, but did not reach its original
volume. For comparison, the cell response at 21°C is also
included in Fig 8. Here, the cell did not show any RVD.
DISCUSSION
Quantitative models describing the deformation and flow
of human neutrophils in the microcirculation require accurate
morphometric parameters such as volume and surface area.
Thus, the precise measurement of neutrophil volume has
been of interest. Using LM and TEM-stereology, SchmidSchonbein et a12 reported a value of I90 pm3, whereas using
micropipette techniques, we recently obtained a value for
cell volume that is 47% larger. The average cell volume for
I50 cells from five individuals, measured during slow aspiration, is 28 1 5 30 fim3.' In this study, using both undeformed
spherical cells and micropipette-deformed cells, we again obtained a value of 294 pm3 36 and 304 f 27 pm3 for the
neutrophil volume at 300 mOsm. In our micropipette measurements, the microvilli contributed negligibly to volume
measurements. Since our values for cell volumes agreed in
both optical and pipette measurements, it is reasonable to
assume that the microvilli did not enhance the apparent cell
diameter, as seen under a light microscope. Our results are
more compatible with the measurement of Evans and Yeung,6
*
1
2
3
4
5
6
Fig 5. Volumes of cells relative to the isotonicvolume, V, plotted
against the reciprocal of the osmolality of the suspending media
relative to the isotonicosmolality, P. The isotonic osmolality is 300
mOsm and the slope of the line corresponds to Ponder's value, R.
(A)Measurements of undeformed spherical cells, and (0)
measurements of deformed cells in pipettes.
who obtained a value of 322 pm3 at 290 mOsm, and also
with in vivo measurements in humans by Bagge and Brinemark.14 The discrepancy between the results of SchmidSchonbein et a12and those presented here and elsewhere could
be that the cells shrunk during the preparation procedures
for TEM." The measurements of Schmid-Schonbein et al
were based on the actual mass of the cell derived from TEM
images. Due to the unavoidable drying shrinkage associated
with SEM images, neutrophils shown in Fig 3e through h are
not the true representation of neutrophil dimensions. In
agreement with Schmid-Schonbein et a1,* we also observed
the loss of membrane foldings for the cells fixed in a swollen
state in hypotonic solutions (Fig 3e). This could be due to
an isotropic shrinkage during critical-point drying.' The cell
shape and surface characteristics are preserved with a uniform
change in volume at every point.
Neutrophils and other leukocytes (in contrast to RBC) have
a large excess surface area because of the membrane foldings
or villi. During swelling under hypotonic conditions, the volume of the cell increases, with a decrease in the number and
Table 2. Neutrophil Surface Areas at Lysis
Area
Area ratio
Initial
Slow Swelling
Fast Swelling
214pm2
1
546 gm2
2.6
456 Fmz
2.1
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NEUTROPHIL VOLUME AND OSMOTIC PROPERTIES
3
2779
1
0
0
f-
Cell lysis
12
n
E
A
d
w
n
11
0
.
10
B
9
solution. the swelling is gradual and the pores formed could
reseal and thus slow down the lysing process. This slow lysing
process could activate the formation of a F-actin network
and fusion of granule and plasma membranes (Fig 7 ) .Membrane transfer from the lysosomal granules to the plasma
membrane has been observed after degranulation.” Both of
the above processes could stiffen and increase the membrane
surface area.
In animal cells. the major determinant of cell volume is
water content, which constitutes approximately 7O40 of the
total cellular mass.’” Cell volume is determined by the content
of osmotically active solutes and the osmolarity of the extracellular fluid. The largest component of the active solutes has
been determined to be inorganic ions: 140-mmol/L K’. 10to 20-mmol/L Na’. and 30- to 50-mmol/L CI-.”.” On this
basis, control of cell volume must depend. to a large extent.
on control of ion content. particularly of K’. Na’. and CI-.
Permeability to these ions under physiologic conditions is
generally much lower than the water permeability. It has been
shown that in Ehrlich ascites tumor cells. Na’. K’. and CIpermeability are all 5 orders of magnitude lower than the
water permeability.” Thus. the membranes of animal cells
can be regarded as semipermeable. Many cell types. including
leukocytes. allow the flow ofwater from one side to the other.
They behave as perfect osmometers. as predicted by the Boyle
Van’t-Hoff relationship. when subjected to changes in the
osmolality of their surrounding medium.’4.” In our studies
with neutrophils at 2 I “C. we showed that human neutrophils
indeed behaved as ideal osmometers (Fig 5). RVD was not
observed over a period of I hour. RVD modulated by temperature has also been reported by Deutsch and Lee.” who
showed that quiescent mouse T-cell clone (L2) did not exhibit
RVD at 17 or 25°C. but show limited RVD at 37°C. However. Grinstein et
reported RVD for human lymphocytes
at 24°C. At 37°C. human neutrophils also showed RVD. as
shown in Fig 8.
The underlying mechanisms in the RVD of cells is at present unclear. It is generally accepted that this regulation is
B
0
50
100
150
200
t (sec)
Fig 6. A neutrophilwas transferred from an isotonic suspending
medium to distilled water. The cell dimensions were monitored before and after the cell Ivsed.
size of the foldings as they are incorporated into the surface
area of the cell.’” These observations suggest that. during
moderate swelling. large stresses are not being imposed on
the bilayer structure of the membrane. After a secondary
shrinking phase (RVD). the membrane foldings reappear. In
distilled water, a large osmotic pressure developed between
the inside and outside of the cell. resulting in large pore formation and water influx. After lysis. the cortical tension that
exists even in passive ~ e l l ~ . l ’could
. ~ * be responsible for bringing the cell back to its initial size (Fig 6). In a 60-mOsm
Fig 7. tight microscopicimage
of neutrophils that were swollen
slowly and finally lysed in a 60mOsm suspending medium.
Granules are seen adheringto the
plasma membranes.
I
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TING-BEALL, NEEDHAM, AND HOCHMUTH
2780
12
0.
0
0
n
0 0
.
37°C
E
3-
v
n
0
20
10
30
t(min)
Fig 8. Volume regulation of human neutrophils at (0)
21 "C and
( 0 )37°C. The cells were transferred from an isotonic suspending
medium to a 100-mOsm hypoosmoticsuspending medium and allowed to equilibrate for 25 minutes.
mediated by independent K' and CI- efflux, which osmotically withdraws water from the cell^.'^,^^.^' However, the signals that trigger K+ and C1- channels during hypotonic stress
are yet to be identified. Many hypotheses have been proposed.
These include Ca2+-activatedK+ channels and stretched-induced C1- channel^.^^,'^^^^ However, accumulating evidence
suggests that intracellular Ca2+ can induce RVD, but this
does not imply that swelling-induced K' permeability is Ca2+activated.12In lymphocytes, neither Ca2+-activatedK+ channels in RVD nor increase in intracellular Ca2+has been obs e r ~ e d . Our
~ ~ ~results
~ ' also indicate that neutrophils showed
RVD at 37°C despite the absence of Ca2+in the isolation
medium and prolonged exposure to Ca2+-freemedium (Fig
8). More sensitive Ca2+fluorescent probes,**which allow accurate minute quantitation of the intracellular free Ca2+level
during RVD, should give us some insight into the role of
ca2+.
REFERENCES
1. Needham D, Hochmuth RM: Rapid flow of passive neutrophils
into a 4 pm pipet and measurement of cytoplasmic viscosity. J Biomech Eng 112:269, 1990
2. Schmid-Schonbein GW, Shih YY, Chien S: Morphometry of
human leukocytes. Blood 56366, 1980
3. Ponder E: Hemolysis and Related Phenomena. New York, NY,
Grune & Stratton, 1948
4. Linderkamp 0, Meiselman HJ: Geometric, osmotic, and
membrane mechanical properties of density-separated human red
cells. Blood 59: 112 I , 1982
5. Needham D, Armstrong M, Hatchell D, Nunn RS: Rapid deformation on passive polymorphonuclear leukocytes: The effects of
pentoxifylline. J Cell Physiol 140:549, 1989
6. Evans E, Yeung A: Apparent viscosity and cortical tension of
blood granulocytes determined by micropipet aspiration. Biophys J
56:151, 1989
7. McGarvey KA, Reidy MA, Roach MR: A quantitative study
of the preparation of rabbit aortic endothelial cells for scanning electron microscopy. J Microsc I 18:229, 1980
8. Hempling HG, Thompson S, Dypre A: Osmotic properties of
human lymphocyte. J Cell Physiol 93:293, 1977
9. McGann LE, Walterson ML, Hogg LM: Light scattering and
cell volumes in osmotically stressed and frozen-thawed cells. Cytometry 9:33, 1988
IO. Rorive G, Gilles R: Intracellular inorganic osmotic effectors,
in Gilles R (ed): Mechanisms of Osmoregulation in Animals,
Maintenance of Cell Volume. New York, NY, Wiley, 1979, p 83
11. Gilles R: Intracellular organic osmotic effectors, in Gilles R
(ed): Mechanisms of Osmoregulation in Animals, Maintenance of
Cell Volume. New York, NY, Wiley, 1979, p 11 1
12. Deutsch C, Lee SC: Cell volume regulation in lymphocytes.
Renal Physiol Biochem 35:260, 1988
13. Grinstein S, Foskett J K Ionic mechanisms of cell volume
regulation in leukocytes. Annu Rev Physiol 52:399, 1990
14. Bagge U, Brlnemark PI: White blood cell rheology. An intravital study in man. Adv Microcirc 7:1, 1977
15. King MW: Dimensional changes in cells and tissues during
specimen preparation for the electron microscope. Cell Biophys 18:
31, 1991
16. Cheung RK, Grinstein S, Dosch HM, Gelfand EW: Volume
regulation by human lymphocytes: Characterization of the ionic basis
for regulatory volume decrease. J Cell Physiol 1 12:189, 1982
17. Tran-Son-Tay R, Needham D, Yeung A, Hochmuth RM:
Time dependent recovery of passive neutrophils after large deformation. Biophys J 60:856, 1991
18. Needham D, Hochmuth RM: A sensitive measure of surface
stress in the resting neutrophil. Biophys J 6 1:1664, 1992
19. Brown WJ, Shannon WA, Snell WJ: Specific and azurophilic
granules from rabbit polymorphonuclear leukocytes. 11. Cell surface
localization of granule membrane and content proteins before and
after degranulation.
20. Loewy AG, Siekevitz P: Cell Structure and Function. Fort
Worth, TX, Holt Rinehart and Winston, 1963
21. Lichtman MA, Jackson AH, Peck WA: Lymphocyte monovalent cation metabolism: Cell volume cation content and cation
transport. J Cell Physiol 80:383, 1972
22. Negendank W: Studies of ions and water in human lymphocytes. Biochim Biophys Acta 694:123, 1982
23. Hoffman EK, Simonsen LO: Membrane mechanisms in volume and pH regulation in vertebrate cells. Physiol Rev 69:3 15, 1989
24. Grinstein S, Rothstein A, Sarkadi B, Gelfand EW: Responses
of lymphocytes to anisotonic media: Volume-regulating behavior.
Am J Physiol 246:C204, 1984
25. Sarkadi B, Parker JC: Activation of ion transport pathways
by changes in cell volume. Biochim Biophys Acta 1071:407, 1991
26. Grinstein S, DuPre A, Rothstein A: Volume regulation by
human lymphocytes, role of calcium. J Am Physiol 79:849, 1982
27. Rink TJ, Sanchez A, Grinstein S, Rothstein A: Volume restoration in osmotically swollen lymphocytes does not involve changes
in free Ca2+concentration. Biochim Biophys Acta 762:593, 1983
28. Grynkiewcz G, Poenie M, Tsien RY: A new generation of
Ca2+indicators with greatly improved fluorescent properties. J Biol
Chem 260:3440, 1985
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1993 81: 2774-2780
Volume and osmotic properties of human neutrophils
HP Ting-Beall, D Needham and RM Hochmuth
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