Proposal for a new evaluation of phagocytosis using different sizes of fluorescent polystyrene microspheres

doi:10.4236/abc.2013.36064

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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: *[email protected]

Received 25 September 2013; revised 26 October 2013; accepted 18 November 2013

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.

OPEN ACCESS

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

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.

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.

R. Enomoto et al. / Advances in Biological Chemistry 3 (2013) 556-563

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.

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

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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