Advances in Biological Chemistry, 2013, 3, 556-563 ABC
http://dx.doi.org/10.4236/abc.2013.36064 Published Online December 2013 (http://www.scirp.org/journal/abc/)
Proposal for a new evaluation of phagocytosis using
different sizes of fluorescent polystyrene microspheres
Riyo Enomoto1,2, Makoto Imamori1, Ayoumi Seon1, Kozue Yoshida1, Aya Furue1, Hirofumi Tsuruda1,
Eibai Lee-Hiraiwa1,2*
1Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan
2Cooperative Research Center of Life Sciences, Kobe Gakuin University, Kobe, Japan
Email: *elee@pharm.kobegakuin.ac.jp
Received 25 September 2013; revised 26 October 2013; accepted 18 November 2013
Copyright © 2013 Riyo Enomoto et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
To investigate phagocytosis, peritoneal-resident and
J774.1 macrophages were incubated with fluorescent
polystyrene microspheres measuring 1.0 μm in diam-
ter at 200 particles per cell. The amount of phago-
cytized microspheres increased with incubation time,
and both cell types had similar phagocytic activity.
Further, we investigated the phagocytosis of different
sizes of microspheres by J774.1 macrophages. To
adequately evaluate phagocytosis, varying amounts of
different sizes of microspheres were added to J774.1
cells, and their phagocytic activities were evaluated.
When the microspheres were added at a density of 20
particles per cell, few small microspheres (<1.0 µm in
diameter) were phagocytized. This result suggested
that their low amount caused difficulty in evaluating
phagocytosis. In contrast, when the same variety of
microspheres was added at a density of 200 particles
per cell, phagocytosis of large microspheres (>3 µm in
diameter) could not be evaluated because of cytotox-
icity. Thus, the amount of different sizes of micro-
spheres added is important for precisely evaluating
phagocytic activity. When the amount of different
sizes of microspheres added was standardized to pro-
vide a set amount of total surface area, phagocytosis
of these microspheres could be adequately evaluated
and compared. To determine the effects of phagocy-
tosis on cell viability and proliferation, cells incubated
with different sizes of microspheres were assayed us-
ing a cell counting kit. We found that phagocytosis
had no effect on cell viability or proliferation and was
independent of particle size. Furthermore, cells al-
ready phagocytized microspheres retained their pha-
gocytic activity.
Keywords: Macrophage; Phagocytosis; Polystyrene
Microsphere; Total Surface Area
1. INTRODUCTION
Dying cells [1-5] or foreign materials [6] are rapidly re-
moved in vivo, both by neighboring tissue cells and
professional phagocytes such as macrophages and den-
dritic cells. Recently, micron-, submicron-, and nano-
scale particles composed of various materials have been
used as delivery devices for living cells or tissues. Most
of these artificial particles are also phagocytized by
macrophages present in tissues or organs throughout the
body [7,8], especially, particles with a negative charge
are recognized via scavenger receptors and phagocytized
by macrophages.
Phagocytosis by macrophages has often been investi-
gated using fluorescent polystyrene microspheres. Ma-
crophage activity is generally evaluated by measuring the
number of phagocytized microspheres. Because the num-
ber of microspheres phagocytized increases in proportion
to the number added to cultures, experimental conditions
are very important in accurately evaluating phagocytic
activity. However, currently, researchers set their own
original criteria for the addition of microspheres and
evaluation of phagocytosis because no standardized con-
ditions for such assays have been determined. This situ-
ation complicates the interpretation and comparison of
related data in many reports concerning phagocytosis.
Currently, microspheres are often added in any amount
or weight per culture area for the evaluation of pha-
gocytosis, but these conditions are presumably unsuitable
for the addition of extremely large or small microspheres
or those having different specific gravities. The recent
developments of new materials and nano-scale tech-
nologies are key components in the field of delivery
devices. Thus, new standardized conditions that are
*Corresponding author.
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R. Enomoto et al. / Advances in Biological Chemistry 3 (2013) 556-563 557
widely applicable are necessary for the evaluation of
phagocytosis. Thus, in this study, we developed new
standardized conditions for the addition of microspheres.
Furthermore, we assessed cell viability and cell activity
of macrophages after phagocytosis using these stan-
dardized conditions.
2. MATERIALS AND METHODS
2.1. Microsphere
Various sizes of Fluoresbrite® yellow–green fluorescent
polystyrene microspheres were purchased from Poly-
sciences Inc. (Warrington, PA). FluoSphere®-red fluo-
rescent 1.0-µm microspheres were purchased from Mole-
cular probes/Life Technologies (Grand Island, NY). Dia-
meter coefficient of variation (CV) of each microsphere
was 3% to 10%. All experiments were conducted with
carboxylate-modified microspheres.
2.2. Cell Culture
Peritoneal resident macrophages were harvested from
12-14-week-old female Wistar rats and suspended in
RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) sup-
plemented with 10% fetal bovine serum (FBS) (Biowest,
Tokyo, Japan). J774.1 macrophages were maintained in
in 100-mm plastic dishes containing the same medium.
When the cells reached 70% - 80% confluence, they
were subcultured and passaged into new 12- or 96-well
plastic plates or chamber slides and precultured at 37˚C
in an atmosphere of 5% CO2 in air.
2.3. Addition of Microspheres to Macrophages
The cells were plated into 12-well plates (8 × 104 cells/
well) and pre-cultured for 2 days (until 70% - 80% con-
fluent, approximately 2 × 105 cells/well). The micro-
spheres were suspended in RPMI 1640 medium con-
taining 10% FBS, and the suspension was added to the
cells. We evaluated phagocytosis using 1.0 μm micro-
spheres (4 × 107 particles/well, 200 particles per cell).
When we measured phagocytosis of other sizes of micro-
spheres, we compensated by adjusting for total volume
or total surface area in relation to the size of the micro-
spheres and amount added. The amount of microspheres
of each size added to the cells is shown in Table 1.
Upper section of Table 1 shows conditions standardized
for volume; the amount of microspheres of each size
added was calculated so that the total volume of added
microspheres of each size was equal to the total volume
of 1.0 μm microspheres used. Lower section of Table 1
shows conditions standardized for surface area. The
amount of microspheres of each size added was calcula-
ted so that the total surface area of added microspheres
of each size was equal to the total surface area of the 1.0
Table 1. Amount ratios of fluorescent microspheres to cells
corrected by volume and surface area.
Diameter (μm)Volume (πμm3) Factor Cell: Microsphere
0.1 0.00017 ×1000 1:200000
0.2 0.0013 ×125 1:25000
0.5 0.021 ×8.0 1:1600
1.0* 0.167 ×1.0 1:200
2.0 1.33 ×0.125 1:25
3.0 4.50 ×0.037 1:7
6.0 36.0 ×0.005 1:1
Diameter (μm)Surface area (πμm2) Factor Cell: Microsphere
0.1 0.01 ×100 1:20000
0.2 0.04 ×25 1:5000
0.5 0.25 ×4.0 1:800
1.0* 1.0 × 1.0 1:200
2.0 4.0 ×0.250 1:50
3.0 9.0 ×0.111 1:22
6.0 36.0 ×0.028 1:6
*We used this value as a base value.
μm microspheres used.
2.4. Quantitative Evaluation of Phagocytosis
The medium was removed from 12-well plates and the
fluorescent microspheres dispersed in growth medium
were added to the cultured cells. The cells were incu-
bated at 37˚C in 5% CO2 for indicated times. At the end
of the incubation period, cells were washed twice with
phosphate buffered saline (PBS) to remove free micros-
pheres and harvested by trypsinization. The harvested
cells were suspended in PBS and analyzed by flow cyto-
metry (FACSCanto, BD Biosciences, Franklin Lakes,
NJ). Flow cytometry data are shown as fluorescent side-
scatter dot plots. We gated each population of cells
according to the amount of microspheres engulfed, from
0 to 4 or more, and calculated the percentage of cells in
each population.
2.5. Morphological Observation of Phagocytosis
Fluorescent microspheres were added to cells in chamber
slides and incubated at 37˚C in an atmosphere of 5%
CO2 for indicated times. At the end of the incubation pe-
riod, cells were washed twice with PBS to remove free
microspheres. Cells on glass chamber slides were di-
rectly observed by confocal laser scanning microscopy
(FV-1000D, Olympus).
2.6. Evaluation of Cytotoxicity and Proliferation
Cells were plated in two 96-well plates (5 × 103 cells/
well) and pre-cultured for 2 days (until approximately
50% confluent). Different sizes of fluorescent micro-
spheres were added to the cells in varying amounts to
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R. Enomoto et al. / Advances in Biological Chemistry 3 (2013) 556-563
558
provide an equivalent total surface area. Cells were
incubated at 37˚C in an atmosphere of 5% CO2 for 3 h.
Cells were washed twice with PBS, and fresh growth
medium was added to the wells. One plate was analyzed
immediately and the other was incubated at 37˚C in an
atmosphere of 5% CO2 for a further 24 h. Cytotoxicity
and proliferation immediately and 24 h after phago-
cytosis were evaluated using Cell Counting Kit-8 (CCK-
8; Dojindo, Kumamoto, Japan).
3. RESULTS AND DISCUSSION
3.1. Phagocytosis of Microspheres by
Macrophages
Phagocytosis involves a series of processes including
binding to the cell surface and uptake by the cell. In our
analysis of cells by flow cytometry, all microspheres
attached to the cell surface or taken up by the cells were
assumed to be phagocytized microspheres. To remove
microspheres nonspecifically adsorbed to the cell surface,
cell suspensions after incubation with fluorescent poly-
styrene microspheres were centrifuged in PBS containing
3% bovine serum albumin (BSA). Microspheres non-
specifically adsorbed to the cell surface separated from
the cells due to difference in density between the free
microspheres and cells. Washing in PBS/BSA solution
did not affect the amount of microspheres phagocytized
by cells (data not shown), but helped wash out micro-
spheres nonspecifically adsorbed to cells. In subsequent
experiments, the cells were washed with PBS. To elu-
cidate whether or not smaller microspheres are phago-
cytized, 0.1-µm microspheres were added to cultures of
the same cells. As shown in Figure 1, both 0.1-µm (4 ×
109 particles/well, 20,000 particles per cell) and 1.0-µm
(4 × 107 particles/well, 200 particles per cell) micro-
spheres were taken up by the cells.
To compare the phagocytic activity of peritoneal resi-
dent and non-stimulated J774.1 macrophages, both types
of cells were incubated with 1.0 μm microspheres (4 ×
107 particles/well, 200 particles per cell). Peritoneal resi-
dent and J774.1 macrophages both phagocytized micro-
spheres and the amount of microspheres phagocytized
increased with incubation time in both cell types. Since
J774.1 macrophages resembled peritoneal resident ma-
crophages in their phagocytic activity (Figure 2), we
used J774.1 macrophages in subsequent experiments.
3.2. Relation of Phagocytosis to the Amount of
Microspheres Added
Different sizes of microspheres were added to J774.1
cells (4 × 106 particles/well, 20 particles per cell), and
their phagocytic activities were evaluated. Few small mi-
crospheres (<1.0 µm in diameter) were phagocytized
(Figure 3), suggesting that small amounts of these mi-
Figure 1. Phagocytosis of J774.1 macro-
phages incubated with different sizes of
microspheres. Cells were incubated with
0.1- or 1.0-µm microspheres according to
lower section of Table 1 for 1 h. Cells
phagocytizing microspheres were observed
from the top (cell surface) to the bottom
using a confocal laser scanning microscope.
Images were acquired every 0.5 µm.
(a)
(b)
Figure 2. Comparison of phagocytosis between J774.1 macro-
phages and rat peritoneal macrophages. (a) Percentages of each
population of macrophages were plotted. Closed circles, cells
engulfing 1 microsphere; open circles, cells phagocytizing 2
microspheres; closed squares, cells phagocytizing 3 microsph-
eres; open squares, cells engulfing four microspheres. (b) Per-
centages of macrophages that phagocytized microspheres were
plotted. Closed triangles, cells phagocytizing nothing; open
train-gle, cells phagocytizing more than 1 microsphere.
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R. Enomoto et al. / Advances in Biological Chemistry 3 (2013) 556-563 559
Figure 3. Effect of the amount of microspheres on phagocyto-
sis in J774.1 macrophages. Cells were cultured in the presence
of different sizes of microspheres for 3 h. All micro-spheres
were added to cells at 20 particles per cell. Cells were washed 3
times and analyzed using a flow cytometer. The data were ana-
lyzed and each population was indicated on X-axis.
crospheres caused difficulty in evaluating phagocytosis.
Therefore, we added these microspheres to the cells un-
der different conditions (4 × 107 particles/well, 200 par-
ticles per cell) (Figure 4). The phagocytosis of micros-
pheres less than 3.0 μm in diameter was able to evaluate
under this condition. But when the microspheres more
than 3.0 μm in diameter were added at this condition
(200 particles per cell), the phagocytosis could not eva-
luate due to these cytotoxicity. These results suggest that
the amount of microspheres added to cells in culture is
important in precisely evaluating phagocytosis. To com-
pensate for differences in the size and amount of micro-
spheres added, microspheres of each size were added to
cells as shown in Table 1. These conditions compensated
for differences in the total volume or total surface area of
microspheres, and results revealed that the phagocytic
activity of cells decreased as the size of the microspheres
increased (Figures 5 and 6). Since cells that engulfed
microspheres of less than 0.5-μm diameter could not be
separated from the remaining cell population, data are
shown as dot plots (Figures 5(b) and 6(b)). Among all
sizes of microspheres added according to upper section
of Table 1, phagocytosis of microspheres of less than 1.0
μm was very high (Figure 5). This phenomenon may be
caused by the addition of too many microspheres. In con-
trast, few microspheres over 4.5-μm diameter were pha-
gocytized (Figure 5(a)). However, these results suggest
that the amount of microspheres is insufficient to eva-
luate phagocytosis. Particles are generally added to cells
as voluntary weight per culture area or per fluid volume
in a phagocytic assay. We consider that this method is
suitable for the addition of particles of equal specific gra-
vity. However, appropriate correction is necessary when
the particles added are of different sizes, specific gra-
vities, or composed of different materials. Among all
sizes of microspheres added according to lower section
of Table 1, phagocytosis of all sizes of microspheres
could be evaluated (Figure 6). Since phagocytosis begins
with the interaction between the surfaces of the micros-
pheres and the cell surface, compensation for total sur-
face area (lower section of Table 1) appears to be a be-
tter method of compensating for the variable amounts of
microspheres in a phagocytic assay. In subsequent expe-
riments, microspheres were added in appropriate a-
mounts to provide the same total surface area.
3.3. Effects of Phagocytosis on Cell Viability and
Cell Function
To elucidate whether or not the phagocytized micro-
spheres had deleterious effects on J774.1 macrophages,
cell viability and proliferation were determined. The in-
tracellular microspheres had no effect on cell viability or
proliferation regardless of microsphere size (Figure 7(a)).
Next, the phagocytic activity of cells having phago-
Figure 4. Effect of the amount of microspheres on phagocyto-
sis in J774.1 macrophages. Cells were cultured in the presence
of different sizes of microspheres for 3 h. All microspheres
were added to cells at 200 particles per cell. Cells were washed
3 times and analyzed using a flow cytometer. The data were
analyzed and each population was indicated on X-axis.
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R. Enomoto et al. / Advances in Biological Chemistry 3 (2013) 556-563
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560
(a)
(b)
(c)
Figure 5. Phagocytosis by J774.1 macrophages when different sizes of microspheres were added at equal total volumes. Cells were
incubated with different sizes of microspheres according to upper section of Table 1 for 1 h. (a and b) Cells were washed 3 times and
analyzed using a flow cytometer. (c) Cells were ob- served using a confocal laser scanning microscope after flow cytometry analysis.
cytized microspheres was measured. Cells phagocytized
0.1 or 1.0 μm green or red microspheres (Figure 7(b)).
Before phagocytosis, the presence of intracellular micro-
spheres did not affect subsequent phagocytic activity and
was independent of microsphere size. J774.1 cells re-
tained phagocytic activity for 24 h after the first pha-
gocytosis. Subsequently, J774.1 cells underwent repeated
cell division along with microspheres, and the number of
microspheres in the cells decreased because of repeated
distribution to cells (data not shown). Although cell death
of macrophages after phagocytosis of silica microspheres
has been reported [9-11], the presence of intracellular
polystyrene microspheres had no effect on cell viability
or phagocytosis, which is the most basic cell function of
macrophages used in this study.
4. CONCLUSION
Our results show that macrophages can phagocytize 0.1 -
6.0-μm polystyrene microspheres, demonstrating the im-
portance of standardizing the amount of microspheres
added to cells when evaluating phagocytosis. The
amount or weight of microspheres added per culture area
in phagocytic assays is often arbitrary. The addition of a
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R. Enomoto et al. / Advances in Biological Chemistry 3 (2013) 556-563 561
(a)
(b)
(c)
Figure 6. Phagocytosis by J774.1 macrophages when different sizes of microspheres were added at equal total surface areas. Cells
were incubated with different sizes of microspheres according to lower section of Table 1 for 1 h. (a and b) Cells were washed 3
times and analyzed using a flow cytometer. (c) Cells were observed using a confocal laser scanning microscope after flow cytometry
analysis.
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R. Enomoto et al. / Advances in Biological Chemistry 3 (2013) 556-563
562
(a)
(b)
Figure 7. Effects of phagocytosis on cell viability, proliferation and phagocytic activity in J774.1 macrophages. (a) Cells were
incubated with or without different sizes of microspheres for 3 h. At the end of incubation, cells were washed and some were
re-incubated in fresh growth medium for 24 h. Cell survival immediately and 24 h after phagocytosis was determined by CCK-8
assay. Open circle, control cell; closed circle, cells incubated with various sizes of microspheres. (b) Cells were incubated with or
without 0.1- or 1-µm green fluorescent microspheres (closed triangles) for 3 h and then washed. Cells were then re-incubated in fresh
growth medium for 24 h after which they were incubated with 1-µm red fluorescent microspheres (open triangles) for 3 h. Cells were
observed by confocal laser scanning microscopy.
given amount of microspheres per culture area seems to
be an orderly method, but this may be inappropriate for
evaluation of phagocytosis of varying sizes of micro-
spheres. On the other hand, the addition of a set weight
per culture area complicates evaluation of phagocytosis
of the same size of microspheres having different spe-
cific gravities. Our suggestion of a standardized method
is better than the traditional methods in the evaluation of
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R. Enomoto et al. / Advances in Biological Chemistry 3 (2013) 556-563 563
phagocytosis. Additionally, the polystyrene microspheres
which are taken up in these conditions have no effect on
cell viability or basic cell function.
5. ACKNOWLEDGEMENTS
This work was supported by the MEXT-Supported Program for the
Strategic Research Foundation at Private Universities, 2012-2017.
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