- Email: [email protected]
[4] In Vitro and in Vivo measurement of phagocytosis by flow cytometry
[4]
183
FLOW CYTOMETRY AND PHAGOCYTOSIS
[4] I n Vitro a n d in Vivo M e a s u r e m e n t of P h a g o c y t o s i s b y Flow Cytometry By
CARLETON C . STEWART, BRUCE
E.
LEHNERT,
and
JOHN A . STEINKAMP
Introduction Many in vitro assays have been developed to quantify the ingestion of opsonin-dependent and opsonin-independent phagocytosis as the overall physiologic importance of phagocytosis has been increasingly recognized. Often, phagocytosis is quantitated microscopically, but this approach is tedious and time consuming, and only a small proportion of the total mononuclear phagocyte population can be evaluated. In this chapter, we describe experimental approaches by which the phagocytic activity of virtually tens of thousands of cells in a population of macrophages can be rapidly measured in vitro on a cell-by-cell basis using flow cytometry. Following a quick, quantitative microscopic confirmation that particles are engulfed and not merely adhered to the surfaces of the macrophages after a particle-cell coincubation period (see this volume [6]), phagocytosis measured by flow cytometry can be quantitatively described in two ways. First, the percentage of cells with engulfed particles can be determined. Second, individual particulate burdens within each cell can be expressed as frequency distributions showing the frequency of phagocytic cells with a given number of internalized particles. The percentage of cells with particles indexes the fraction of cells doing the work, whereas the particle-cell distribution data serve to index the extent of phagocytic work performed by each phagocytic macrophage. Methods In Vitro Assay Cell Populations. Methodologies for obtaining mononuclear phagocytes from the bone marrow, 2 peripheral blood, 3 peritoneal cavity, 4 pleui j. A. Steinkamp, J. S. Wilson, G. C. Saunders, and C. C. Stewart, Science 215, 64 (1982). 2 C. C. Stewart, in "Methods for Studying Mononuclear Phagocytes" (D. O. Adams, P. J. Edelson, and H. S. Koren, eds.), p. 5. Academic Press, New York, 1981. 3 C. C. Stewart, in "Methods for Studying Mononuclear Phagocytes" (D. O. Adams, P. J. Edelson, and H. S. Koren, eds.), p. 21. Academic Press, New York, 1981. 4 R. E. Conrad, in "Manual of Macrophage Methodology" (H. B. Herscowitz, H. T. Holden, J. A. Bellanti, and A. Ghaffar, eds.), p. 5. Dekker, New York, 1981.
METHODSIN ENZYMOLOGY,VOL. 132
Copyright© 1986by AcademicPress, Inc. All rightsof reproductionin any formreserved.
184
SPECIAL METHODS FOR MEASURING PHAGOCYTOSIS
[4]
ral space, 5 lymph nodes, 6 and the lung 7 have all been previously described in great detail. We refer to the cited publications for these procedures (see also this series, Vol. 108 [9], [25], [26], [27], [28], and [29]). Media and Test Particles. Cell populations should be maintained in a complete medium that is buffered with an organic buffer and not with sodium bicarbonate. Macrophages are exquisitely sensitive to a pH exceeding 7.4. When a bicarbonate buffered medium is used, the appropriate pH cannot be maintained in the absence of a continuous atmosphere supplemented with 5 to 10% carbon dioxide. This problem can be circumvented by using an organic buffer such as HEPES or MOPS (10 to 20 mM). The medium we use is a-minimum essential medium supplemented with 20 mM 2-N-morpholinopropanesulfonic acid and 10% fetal bovine serum (a-10 MOPS). The osmolality of the medium is adjusted with sodium chloride to 300 mOsm and the pH is adjusted to 7.2. Another reagent used is the same medium containing 2% bovine serum albumin (fraction V, Sigma Chemical Company, St. Louis, MO). This medium is used to separate free test particles from the cells following the particle-macrophage coincubation phase of the assay. For test particles, we recommend using fluorescent microspheres from Polysciences, Inc. (Paul Valley Industrial Park, Warrington, PA). These polystyrene microspheres are available in different fluorescent colors over a range of sizes. Depending on the fluorescent color, the excitation wavelength will range from the ultraviolet to the red (see the table). For the phagocytic assay, it is important to use 1.5- to 2.0-/~m-diameter microspheres because smaller particles may be pinocytosed by cells other than macrophages. For example, 0.8-/.Lm-diameter microspheres are avidly pinocytosed by fibroblasts and some tumor cell types, but 1.5-/zm-diameter microspheres are not. Also, it is important that the coefficient of variation (CV) [i.e., (standard deviation/mean)100%] of fluorescence of these spheres be less than 2% if the number of particles per cell is to be resolved by flow cytometry. With such a low CV, it is possible to resolve up to five particles per cell. Assay System. The cell suspension is adjusted to I × 106 cells/ml and 3 ml is placed in a 50-ml polypropylene conical centrifuge tube (Falcon No. 2070, Becton Dickinson Labware, Oxnard, CA) and warmed to 37°. The large size of these tubes provides a high surface area to fluid volume ratio suitable for maintaining cells and microspheres in suspension during continuous mixing. Moreover, the polypropylene tubes provide a non5 A. Zlotnik, A. Vatter, R. L. Hayes, E. Blumenthal, and A. J. Crowle, RES: J. Reticuloendothel. Soc. 31, 207 (1982). 6 D. E. Bice, D. L. Harris, C. T. Schnizlein, and J. L. Mauderly, Drug Chem. Toxicol. 2, 35 (1979). 7 B. E. Lehnert and J. Ferin, RES: J. Reticuloendothel. Soc. 33, 293 (1983).
[4]
FLOW CYTOMETRY AND PHAGOCYTOSIS
185
FLUORESCENT MICROSPHERES FOR USE IN FCM PHAGOCYTOSIS EXPERIMENTS AND LASER EXCITATION
Polysciences catalog n u m b e r 17686 17686 17686 17687 17687 17687 17688" 17688" 17689"
Absorbed dye excitation maximum (h) 365 365 365 458 458 458 554 554 686
nm nm nm nm nm nm nm nm nm
Laser/excitation wavelength (h)
Typical fluorescence measurement barrier filter
HeCd/325 nm Argon/333-363 nm Krypton/337-356 nm HeCd/441 nm Argon/457 nm Krypton/406-415 nm Argon/528 nm Krypton/530 nm Krypton/647 nm
GG400 GG400 GG400 GG475 GG495 GG455 OG570 OG570 RG695
" These microspheres also can be excited using an argon- or krypton-pumped CW dye laser.
wettable surface which further retards cell adherence; macrophages adhere to the surface of these tubes at a rate of only 10% per hour even when they are not agitated. The microspheres of a desired color are suspended in culture medium at a concentration of 1 x 108/50 ~1 and sonicated (Ultrasonic Cleaner, Fischer Scientific, Medford, MA) at 37 ° for 30 sec in order to dissociate aggregates of the test particles. Immediately thereafter, 3 x 108 of the microspheres are added to the 3 ml of the cell suspension. With a 100 : 1 particle-to-cell ratio, phagocytosis is not limited by the availability of microspheres over the course of the study. The cells and particles are coincubated at 37° for 30-60 min, during which time a wellmixed system is maintained with a rotating wheel (Multi-Purpose Rotator, Scientific Industries, Inc., Springfield, MA). At desired times during this incubation period, cells can be sampled for kinetic studies of phagocytosis. During the incubation period, 2 ml of the medium containing 2% BSA is put in a 15-ml conical centrifuge tube (Corning No. 25310, Corning Glass Works, Corning, NY). One of these tubes is prepared for each of the suspensions that is being incubated. After the desired time of incubation, the cell-particle suspension is carefully layered on top of the BSA medium and each tube is centrifuged for 10 min at 150 g. The supernatants are aspirated and the cell pellet is resuspended in 3 ml culture medium. This latter centrifugation step separates free particles from the cells, as well as serving to strip loosely adherent microspheres from the ceils' surfaces. After this procedure, the cells are ready for analysis by flow cytometry.
186
SPECIAL METHODS FOR MEASURING PHAGOCYTOSIS
[4]
In Vivo Phagocytosis
It is also possible to quantitate in vivo phagocytosis. Microspheres can be injected intraperitoneally to measure phagocytosis by peritoneal macrophages, injected in the pleural space to measure phagocytosis by pleural macrophages, or administered intravenously to measure phagocytosis by blood monocytes and granulocytes. For all injections, a suspension of 1 z 108 microspheres in 0.25 ml is used. Microspheres can also be instilled into the lungs to measure phagocytosis by alveolar macrophages. For studies of the phagocytic activity of alveolar macrophages in vivo, rats are anesthetized with Ethrane (enflurane, Airco, Madison, WI) and the animals are intrabronchially instilled with 2 x 108 particles in 0.5 ml normal saline using a blunt, 18-gauge needle tipped with a 20-gauge Teflon tube which is advanced just proximal to the carina. After the instillations, the animals are held vertically for 30 sec before being returned to their cages. At selected times thereafter, animal sacrifices are initiated with intraperitoneal injections of 50 mg pentobarbital sodium. Prior to the onset of apnea, the animals are exsanguinated via the carotid arteries. The trachea is then cannulated with an 18-gauge blunt needle secured with a ligature, the lungs and trachea are excised en bloc, and the heart and esophagus are removed. Bronchoalveolar lavage is initiated by instilling 6 ml of room-temperature, divalent cation-free, phosphate-buffered saline (pH 7.2) into the lungs and aspirating the lavage fluid during gentle lung massage. This procedure is repeated an additional seven times. The retrieved lavage fluids (generally >95% of the instilled volume) are pooled in a centrifuge tube maintained in ice. Aliquots of the cell suspensions are layered over newborn bovine serum and centrifuged at 360 g for 10 min (4°) to remove nonphagocytized microspheres. Following removal of the supernatants, the cells are vortexed and resuspended in 0 ° culture medium until analyzed in the flow cytometer. By measuring phagocytosis at several sequential time points after instillation of the microspheres, the kinetics of particle phagocytosis following particle deposition in the lung and intrapulmonary, particle-macrophage relationships over the course of alveolar clearance can be studied. Concurrent with these studies, suspensions prepared from the thoracic lymph nodes can be analyzed for evidence of particle migration from the lungs to these sites. 8 It should be pointed out that two different types of fluorescent microspheres may be used in the in vitro and in vivo phagocytic assays. We have applied this technique, for example, to determine if alveolar macros S.
Hyler, Y. E. Valdez, and B. E. Lehnert,
Toxicologist 5, 180 (1985).
[4]
FLOW CYTOMETRY AND PHAGOCYTOSIS
187
phages that had not phagocytosed microspheres in vivo were also not phagocytic in vitro, or whether they simply did not encounter particles in the lung. ~ If microspheres of one color are instilled and after 2 hr the population is lavaged and then incubated with microspheres of a second color using the in vitro procedure, the maximum phagocytic activity of cells can be measured. With this approach, we found that macrophages which had not phagocytosed in vivo were capable of avid phagocytosis in vitro. Such results suggest the instilled particles were not evenly distributed in the alveoli and, consequently, the microspheres were not available to all members of the alveolar macrophage population in situ. Flow Cytometry (see also this series, Vol. 108 [19])
The cells are analyzed for narrow angle light scatter (cell size) and for fluorescence (phagocytized spheres) as they flow through a flow cell 9 and intersect a laser beam of exciting light (e.g., 457 nm wavelength, argonion laser). 1 Optical sensors measure light scattering and fluorescence on a cell-by-cell basis and the signals are stored in the list mode data format in a computer. 10,1, The data are then reprocessed and displayed as single- or two-parameter cell-size and fluorescence frequency distribution histograms. Typical Results
Typical flow cytometric data obtained with alveolar macrophages harvested 2 hr after an intrabronchial instillation of the microspheres are shown in Fig. 1A and B. Such results illustrate the types of data obtained with either the in vitro or in vivo phagocytic assay. Figure 1A shows the size distribution of lavage-free cells from a Sprague-Dawley rat instilled with 1.83-/xm-diameter green fluorescent spheres. Region 1 represents polymorphonucleated leukocytes (PMN) and lymphocytes. Pulmonary alveolar macrophages that have or have not phagocytized microspheres are contained in region 2. Since the free nonphagocytized spheres are smaller than cells, they do not appear in the cell-size distribution and they are totally discriminated against. Thus, by requiring the fluorescence signals to be in coincidence with the light scattering signals from cells, only cells that have associated spheres are contained in the fluorescence distribution (see Fig. 1B). Data from the fluorescence and size distributions can be used to determine the percentage of cells containing one or more spheres and the 9 j. A. Steinkamp, Rev. Sci. Instrum. 55, 1375 (1984). 1o R. D. Hiebert, J. H. Jett, and G. C. Salzman, Cytometry 1, 337 (1981). H G. C. Salzman, S. F. Wilkins, and J. A. Whitfill, Cytometry 1, 325 (1981).
i
O~-
~.u~
I
I
I
J
+ o °
e~
E~
Z
•
I
.-x.
¢~ m,~
-
+.,,+
+>+. ~
1
~
° SI"13:) 4 0 ~ ] g W N N
~+m
m
[4]
FLOW CYTOMETRY AND PHAGOCYTOSIS
189
A I---- Region II --I
tII) LU O
Log 2 ° Light Scatter
Green Fluorescence
-Q
E Z ¢)
B •
C
Singlets
o m
13
Doublets
n¢
Triilets
Green Fluorescence
Green Fluorescence
FIG. 2. (A) Log 2° light scattering and green fluorescence histograms obtained with a modal sample obtained 30 days after particle deposition in the lung. Left histogram: Region I of the light scatter histogram contains free particles, erythrocytes, and debris, while region II contains the nucleated cells. Right histogram: Fluorescence microspheres associated with the left light scatter distribution. (B) Distribution of single particles and aggregates of two or more microspheres is resolved when the correlated list mode data were reprocessed to obtain fluorescent events associated with events in region I of the light scatter histogram. The ordinate scale of the green fluorescent histogram has been expanded relative to that in (A) for the purpose of demonstration. (C) Distribution of microspheres that are associated with the cells in region II of the above light scatter histogram. The ordinate scale of the green fluorescent histogram has been expanded equivalently to that in (B).
percentage of cells having ingested one to five spheres, or more than five spheres. By dividing the total number of macrophages having phagocytized one or more spheres by the number of cells within region 2 (Fig. 1A--macrophages), the percentage of the cells that phagocytized the particles can be determined. This can be further expanded into the percentage of phagocytic alveolar macrophages containing one, two, three, four, five, and more than five spheres by dividing the total number of phagocytic cells into the number containing one, two, etc., spheres. Sorted cells containing one to five microspheres are shown in Fig. 1C-G.
190
SPECIAL METHODS FOR MEASURING PHAGOCYTOSIS
[4]
Figure 2A-C illustrates the flow cytometric approach we recently used 8 to quantitate the migration of 1.9-/xm-diameter fluorescent (green) microspheres to the thoracic lymph nodes after 4 x 108 of these particles were deposited in the lungs of rats. Cells and particles liberated from the nodes after dissection were prepared in suspension and they were excited by the 488-nm line of an argon laser. Log 2° light scattering and green fluorescence over a 515- to 545-nm wavelength range were measured. Signal processing electronics were set to trigger on any incoming event, i.e., no coincidence requirements. Data were acquired for 30,000 events per sample and stored in the correlated list mode fashion.11 In order to estimate the total number of particles associated with the nodal cells in each analyzed sample, the integrated sum of fluorescent events coincident with region II of the log 2° light scattering histogram, as illustrated in Fig. 2A and C, was determined. Those particulate events falling in the window range for ->4 particles were assigned a minimum value of 4 for computational purposes. This sum was expressed as a fraction of the total number of cells analyzed in region II. This fraction, in conjunction with the total cell yield from the nodes of a given animal, was then used to determine the total numbers of particles associated with all the cells harvested. As with the alveolar macrophages, the particulate burdens in each nodal phagocyte were also determined. The total number of free or non-cell-associated particles in the nodal samples was determined by analyzing region I of the log 2 ° light scatter histogram, Fig. 2A, for the fluorescent particulate events shown in Fig. 2B. The total number of fluorescent particles coincident with region I were expressed as a fraction of the total number of cellular events occurring in region II, and this fraction, in turn, was used in conjunction with nodal cell yields to determine the total number of "free" particles harvested from the lymph nodes. Comments
We have described experimental techniques whereby phagocytosis by mononuclear phagocytes can be rapidly and accurately measured using multiparameter flow cytometry. Quantitative indices of phagocytosis obtained using the flow cytometric approach include the percentage of cells that actually phagocytose particles, and the distribution of particles in the cells that contain them. Therefore, the relative number of cells in a population that has performed the work of phagocytosis, as well as an index of the extent of the work performed by the phagocytic cells, is precisely obtained. Moreover, the in vitro suspension assay system outlined here
[4]
FLOW CYTOMETRY AND PHAGOCYTOSIS
191
allows one to obtain these quantitative indices of phagocytosis by evaluating the phagocytic activity of virtually every cell in an experimental population, unlike some conventional phagocytic assay systems that use monolayers of mononuclear phagocytes formed with adherent subpopulations only. As well, performance of the phagocytic assay with cells and particles in a well-mixed system negates the possibility that the phagocytic activities of the cells may be altered by adherence to a stationary substrate. 12 Furthermore, removal of adherent cells from a substrate is accompanied by death of a large proportion of the cells. The phagocytic assay presented in this chapter, by the nature of the test particles used, measures "nonspecific" phagocytosis. However, since the fluorescent microspheres can be obtained with carboxyl groups, it may be possible to suitably coat them with appropriate opsonins for studying specific receptor-mediated phagocytosis. The finding by van Oss and co-workers that IgG adsorption onto polystyrene spheres augments their phagocytosis by granulocytesl3 lends tentative support for this possibility. Other investigators have used fluorescently labeled biotic particles such as bacteria in conjunction with flow cytometry to measure specific receptor-mediated phagocytosis. 14,15The high coefficient of variation in the fluorescence from these biological test particles, however, restricts their usefulness. The percentage of cells that phagocytize can be determined, but individual cell burdens of particles cannot presently be resolved. Regardless, the microsphere assay that employs unopsonized particles may be especially appropriate for measuring the phagocytic activities of cells like the alveolar macrophages, inasmuch as these phagocytes routinely encounter particles deposited in the alveolar microenvironment, which is relatively deficient in proteins and opsonins.16 In summary, we know of no other automated method, except for the flow cytometric technique described here, that can measure the percentage of a macrophage population that has phagocytized, and also gives discrete information on particulate burdens in individual cells. The flow cytometric approach offers major advantages over existing, conventional techniques. Chiefly, data can be easily obtained from large sample sizes and, because of the rapidity of the method, multiple cell samples can be evaluated over a short period of time. 12 B. E. Lehnert and P. E. Morrow, Immunol. Commun. 13, 313 (1984). t3 C. J. van Oss and M. W. Stenson, RES: J. Reticuloendothel. Soc. 8, 397 (1970). 14 p. Szejda, J. W. Parce, M. S. Seeds, and D. A. Boss, J. Immunol. 133, 3303 (1984). 15 C. F. Bassoe, O. D. Laerum, J. Glette, G. Hopen, B. Haneberg, and C. O. Solberg, Cytometry 4, 254 (1983). 16 H. Y. Reynolds and H. N. Newhall, J. Lab. Clin. Med. 84, 559 (1974).
192
SPECIAL METHODS
FOR MEASURING
PHAGOCYTOSIS
[5]
Acknowledgments This work was supported in part by the Department of Energy, Office of Health and Environmental Research, and the Los Alamos National Flow Cytometry and Sorting Research Resource funded by the Division of Research Resources of NIH (Grant No. P41-RK 01315-02).
[5] O i l - D r o p l e t M e t h o d for M e a s u r i n g P h a g o c y t o s i s
By THOMAS P. STOSSEL Introduction The basis of the oil-droplet method to assay phagocytosis is to stabilize particles containing a marker dye or radioactive compound, feed them to phagocytes, separate uningested particles from the cells by differential centrifugation, extract the marker from the cells, and measure it. The rate of association of the marker with the centrifuged cell pellet as a function of time reflects the rate of phagocytosis. Since its initial description ~the oil-droplet method has been modified in many ways. Originally, the dye oil red O was incorporated as a marker into heavy paraffin oil. Subsequently, diisodecyl phthalate was found to have a more suitable density than paraffin oil. Other modifications have included the use of radioactive lipids as markers 2 and of spin-labeled cholestanone, which can be quenched by ascorbic acid, permitting assessment of the extent to which particles are either adherent to the cells or are incorporated within unsealed vacuoles. Extracellular particles or particles in unsealed vacuoles are quenched whereas particles within closed vacuoles a r e n o t . 3,4 Double-label experiments have been performed by feeding cells particles labeled with either oil red O or perylene, measuring the former spectrophotometrically and the latter fluorimetrically.5 The method has several advantages. The most important one is the fact that the density of suitable oil droplets is such that the efficiency of separation of uningested particles from phagocytes is very high; the ability to remove adherent particles markedly facilitates the precise quantitation of phagocytic rates. By varying the material utilized to stabilize the particles, the recognition mechanisms of phagocytosis can be studied. J T. P. Stossel, R. J. Mason, J. H. Hartwig, and M. Vaughan, J. Clin. Invest. 51, 615 (1972). 2 A. Forsgren, D. Schmeling, and D. Zettervall, Immunology 32, 491 (1977). 3 M. J. Ueda, T. Ito, S.-I. Ohnishi, and T. S. Okada, J. Cell Sci. 51, 173 (1981). 4 T. Ito, M. J. Ueda, T. S. Okada, and S.-I. Ohnishi, J. Cell Sci. 51, 189 (1981). 5 R. D. Berlin, J. P. Fera, and J. R. Pfeiffer, J. Clin. Invest. 63, 1137 (1979).
METHODS IN ENZYMOLOGY, VOL. 132
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
183
FLOW CYTOMETRY AND PHAGOCYTOSIS
[4] I n Vitro a n d in Vivo M e a s u r e m e n t of P h a g o c y t o s i s b y Flow Cytometry By
CARLETON C . STEWART, BRUCE
E.
LEHNERT,
and
JOHN A . STEINKAMP
Introduction Many in vitro assays have been developed to quantify the ingestion of opsonin-dependent and opsonin-independent phagocytosis as the overall physiologic importance of phagocytosis has been increasingly recognized. Often, phagocytosis is quantitated microscopically, but this approach is tedious and time consuming, and only a small proportion of the total mononuclear phagocyte population can be evaluated. In this chapter, we describe experimental approaches by which the phagocytic activity of virtually tens of thousands of cells in a population of macrophages can be rapidly measured in vitro on a cell-by-cell basis using flow cytometry. Following a quick, quantitative microscopic confirmation that particles are engulfed and not merely adhered to the surfaces of the macrophages after a particle-cell coincubation period (see this volume [6]), phagocytosis measured by flow cytometry can be quantitatively described in two ways. First, the percentage of cells with engulfed particles can be determined. Second, individual particulate burdens within each cell can be expressed as frequency distributions showing the frequency of phagocytic cells with a given number of internalized particles. The percentage of cells with particles indexes the fraction of cells doing the work, whereas the particle-cell distribution data serve to index the extent of phagocytic work performed by each phagocytic macrophage. Methods In Vitro Assay Cell Populations. Methodologies for obtaining mononuclear phagocytes from the bone marrow, 2 peripheral blood, 3 peritoneal cavity, 4 pleui j. A. Steinkamp, J. S. Wilson, G. C. Saunders, and C. C. Stewart, Science 215, 64 (1982). 2 C. C. Stewart, in "Methods for Studying Mononuclear Phagocytes" (D. O. Adams, P. J. Edelson, and H. S. Koren, eds.), p. 5. Academic Press, New York, 1981. 3 C. C. Stewart, in "Methods for Studying Mononuclear Phagocytes" (D. O. Adams, P. J. Edelson, and H. S. Koren, eds.), p. 21. Academic Press, New York, 1981. 4 R. E. Conrad, in "Manual of Macrophage Methodology" (H. B. Herscowitz, H. T. Holden, J. A. Bellanti, and A. Ghaffar, eds.), p. 5. Dekker, New York, 1981.
METHODSIN ENZYMOLOGY,VOL. 132
Copyright© 1986by AcademicPress, Inc. All rightsof reproductionin any formreserved.
184
SPECIAL METHODS FOR MEASURING PHAGOCYTOSIS
[4]
ral space, 5 lymph nodes, 6 and the lung 7 have all been previously described in great detail. We refer to the cited publications for these procedures (see also this series, Vol. 108 [9], [25], [26], [27], [28], and [29]). Media and Test Particles. Cell populations should be maintained in a complete medium that is buffered with an organic buffer and not with sodium bicarbonate. Macrophages are exquisitely sensitive to a pH exceeding 7.4. When a bicarbonate buffered medium is used, the appropriate pH cannot be maintained in the absence of a continuous atmosphere supplemented with 5 to 10% carbon dioxide. This problem can be circumvented by using an organic buffer such as HEPES or MOPS (10 to 20 mM). The medium we use is a-minimum essential medium supplemented with 20 mM 2-N-morpholinopropanesulfonic acid and 10% fetal bovine serum (a-10 MOPS). The osmolality of the medium is adjusted with sodium chloride to 300 mOsm and the pH is adjusted to 7.2. Another reagent used is the same medium containing 2% bovine serum albumin (fraction V, Sigma Chemical Company, St. Louis, MO). This medium is used to separate free test particles from the cells following the particle-macrophage coincubation phase of the assay. For test particles, we recommend using fluorescent microspheres from Polysciences, Inc. (Paul Valley Industrial Park, Warrington, PA). These polystyrene microspheres are available in different fluorescent colors over a range of sizes. Depending on the fluorescent color, the excitation wavelength will range from the ultraviolet to the red (see the table). For the phagocytic assay, it is important to use 1.5- to 2.0-/~m-diameter microspheres because smaller particles may be pinocytosed by cells other than macrophages. For example, 0.8-/.Lm-diameter microspheres are avidly pinocytosed by fibroblasts and some tumor cell types, but 1.5-/zm-diameter microspheres are not. Also, it is important that the coefficient of variation (CV) [i.e., (standard deviation/mean)100%] of fluorescence of these spheres be less than 2% if the number of particles per cell is to be resolved by flow cytometry. With such a low CV, it is possible to resolve up to five particles per cell. Assay System. The cell suspension is adjusted to I × 106 cells/ml and 3 ml is placed in a 50-ml polypropylene conical centrifuge tube (Falcon No. 2070, Becton Dickinson Labware, Oxnard, CA) and warmed to 37°. The large size of these tubes provides a high surface area to fluid volume ratio suitable for maintaining cells and microspheres in suspension during continuous mixing. Moreover, the polypropylene tubes provide a non5 A. Zlotnik, A. Vatter, R. L. Hayes, E. Blumenthal, and A. J. Crowle, RES: J. Reticuloendothel. Soc. 31, 207 (1982). 6 D. E. Bice, D. L. Harris, C. T. Schnizlein, and J. L. Mauderly, Drug Chem. Toxicol. 2, 35 (1979). 7 B. E. Lehnert and J. Ferin, RES: J. Reticuloendothel. Soc. 33, 293 (1983).
[4]
FLOW CYTOMETRY AND PHAGOCYTOSIS
185
FLUORESCENT MICROSPHERES FOR USE IN FCM PHAGOCYTOSIS EXPERIMENTS AND LASER EXCITATION
Polysciences catalog n u m b e r 17686 17686 17686 17687 17687 17687 17688" 17688" 17689"
Absorbed dye excitation maximum (h) 365 365 365 458 458 458 554 554 686
nm nm nm nm nm nm nm nm nm
Laser/excitation wavelength (h)
Typical fluorescence measurement barrier filter
HeCd/325 nm Argon/333-363 nm Krypton/337-356 nm HeCd/441 nm Argon/457 nm Krypton/406-415 nm Argon/528 nm Krypton/530 nm Krypton/647 nm
GG400 GG400 GG400 GG475 GG495 GG455 OG570 OG570 RG695
" These microspheres also can be excited using an argon- or krypton-pumped CW dye laser.
wettable surface which further retards cell adherence; macrophages adhere to the surface of these tubes at a rate of only 10% per hour even when they are not agitated. The microspheres of a desired color are suspended in culture medium at a concentration of 1 x 108/50 ~1 and sonicated (Ultrasonic Cleaner, Fischer Scientific, Medford, MA) at 37 ° for 30 sec in order to dissociate aggregates of the test particles. Immediately thereafter, 3 x 108 of the microspheres are added to the 3 ml of the cell suspension. With a 100 : 1 particle-to-cell ratio, phagocytosis is not limited by the availability of microspheres over the course of the study. The cells and particles are coincubated at 37° for 30-60 min, during which time a wellmixed system is maintained with a rotating wheel (Multi-Purpose Rotator, Scientific Industries, Inc., Springfield, MA). At desired times during this incubation period, cells can be sampled for kinetic studies of phagocytosis. During the incubation period, 2 ml of the medium containing 2% BSA is put in a 15-ml conical centrifuge tube (Corning No. 25310, Corning Glass Works, Corning, NY). One of these tubes is prepared for each of the suspensions that is being incubated. After the desired time of incubation, the cell-particle suspension is carefully layered on top of the BSA medium and each tube is centrifuged for 10 min at 150 g. The supernatants are aspirated and the cell pellet is resuspended in 3 ml culture medium. This latter centrifugation step separates free particles from the cells, as well as serving to strip loosely adherent microspheres from the ceils' surfaces. After this procedure, the cells are ready for analysis by flow cytometry.
186
SPECIAL METHODS FOR MEASURING PHAGOCYTOSIS
[4]
In Vivo Phagocytosis
It is also possible to quantitate in vivo phagocytosis. Microspheres can be injected intraperitoneally to measure phagocytosis by peritoneal macrophages, injected in the pleural space to measure phagocytosis by pleural macrophages, or administered intravenously to measure phagocytosis by blood monocytes and granulocytes. For all injections, a suspension of 1 z 108 microspheres in 0.25 ml is used. Microspheres can also be instilled into the lungs to measure phagocytosis by alveolar macrophages. For studies of the phagocytic activity of alveolar macrophages in vivo, rats are anesthetized with Ethrane (enflurane, Airco, Madison, WI) and the animals are intrabronchially instilled with 2 x 108 particles in 0.5 ml normal saline using a blunt, 18-gauge needle tipped with a 20-gauge Teflon tube which is advanced just proximal to the carina. After the instillations, the animals are held vertically for 30 sec before being returned to their cages. At selected times thereafter, animal sacrifices are initiated with intraperitoneal injections of 50 mg pentobarbital sodium. Prior to the onset of apnea, the animals are exsanguinated via the carotid arteries. The trachea is then cannulated with an 18-gauge blunt needle secured with a ligature, the lungs and trachea are excised en bloc, and the heart and esophagus are removed. Bronchoalveolar lavage is initiated by instilling 6 ml of room-temperature, divalent cation-free, phosphate-buffered saline (pH 7.2) into the lungs and aspirating the lavage fluid during gentle lung massage. This procedure is repeated an additional seven times. The retrieved lavage fluids (generally >95% of the instilled volume) are pooled in a centrifuge tube maintained in ice. Aliquots of the cell suspensions are layered over newborn bovine serum and centrifuged at 360 g for 10 min (4°) to remove nonphagocytized microspheres. Following removal of the supernatants, the cells are vortexed and resuspended in 0 ° culture medium until analyzed in the flow cytometer. By measuring phagocytosis at several sequential time points after instillation of the microspheres, the kinetics of particle phagocytosis following particle deposition in the lung and intrapulmonary, particle-macrophage relationships over the course of alveolar clearance can be studied. Concurrent with these studies, suspensions prepared from the thoracic lymph nodes can be analyzed for evidence of particle migration from the lungs to these sites. 8 It should be pointed out that two different types of fluorescent microspheres may be used in the in vitro and in vivo phagocytic assays. We have applied this technique, for example, to determine if alveolar macros S.
Hyler, Y. E. Valdez, and B. E. Lehnert,
Toxicologist 5, 180 (1985).
[4]
FLOW CYTOMETRY AND PHAGOCYTOSIS
187
phages that had not phagocytosed microspheres in vivo were also not phagocytic in vitro, or whether they simply did not encounter particles in the lung. ~ If microspheres of one color are instilled and after 2 hr the population is lavaged and then incubated with microspheres of a second color using the in vitro procedure, the maximum phagocytic activity of cells can be measured. With this approach, we found that macrophages which had not phagocytosed in vivo were capable of avid phagocytosis in vitro. Such results suggest the instilled particles were not evenly distributed in the alveoli and, consequently, the microspheres were not available to all members of the alveolar macrophage population in situ. Flow Cytometry (see also this series, Vol. 108 [19])
The cells are analyzed for narrow angle light scatter (cell size) and for fluorescence (phagocytized spheres) as they flow through a flow cell 9 and intersect a laser beam of exciting light (e.g., 457 nm wavelength, argonion laser). 1 Optical sensors measure light scattering and fluorescence on a cell-by-cell basis and the signals are stored in the list mode data format in a computer. 10,1, The data are then reprocessed and displayed as single- or two-parameter cell-size and fluorescence frequency distribution histograms. Typical Results
Typical flow cytometric data obtained with alveolar macrophages harvested 2 hr after an intrabronchial instillation of the microspheres are shown in Fig. 1A and B. Such results illustrate the types of data obtained with either the in vitro or in vivo phagocytic assay. Figure 1A shows the size distribution of lavage-free cells from a Sprague-Dawley rat instilled with 1.83-/xm-diameter green fluorescent spheres. Region 1 represents polymorphonucleated leukocytes (PMN) and lymphocytes. Pulmonary alveolar macrophages that have or have not phagocytized microspheres are contained in region 2. Since the free nonphagocytized spheres are smaller than cells, they do not appear in the cell-size distribution and they are totally discriminated against. Thus, by requiring the fluorescence signals to be in coincidence with the light scattering signals from cells, only cells that have associated spheres are contained in the fluorescence distribution (see Fig. 1B). Data from the fluorescence and size distributions can be used to determine the percentage of cells containing one or more spheres and the 9 j. A. Steinkamp, Rev. Sci. Instrum. 55, 1375 (1984). 1o R. D. Hiebert, J. H. Jett, and G. C. Salzman, Cytometry 1, 337 (1981). H G. C. Salzman, S. F. Wilkins, and J. A. Whitfill, Cytometry 1, 325 (1981).
i
O~-
~.u~
I
I
I
J
+ o °
e~
E~
Z
•
I
.-x.
¢~ m,~
-
+.,,+
+>+. ~
1
~
° SI"13:) 4 0 ~ ] g W N N
~+m
m
[4]
FLOW CYTOMETRY AND PHAGOCYTOSIS
189
A I---- Region II --I
tII) LU O
Log 2 ° Light Scatter
Green Fluorescence
-Q
E Z ¢)
B •
C
Singlets
o m
13
Doublets
n¢
Triilets
Green Fluorescence
Green Fluorescence
FIG. 2. (A) Log 2° light scattering and green fluorescence histograms obtained with a modal sample obtained 30 days after particle deposition in the lung. Left histogram: Region I of the light scatter histogram contains free particles, erythrocytes, and debris, while region II contains the nucleated cells. Right histogram: Fluorescence microspheres associated with the left light scatter distribution. (B) Distribution of single particles and aggregates of two or more microspheres is resolved when the correlated list mode data were reprocessed to obtain fluorescent events associated with events in region I of the light scatter histogram. The ordinate scale of the green fluorescent histogram has been expanded relative to that in (A) for the purpose of demonstration. (C) Distribution of microspheres that are associated with the cells in region II of the above light scatter histogram. The ordinate scale of the green fluorescent histogram has been expanded equivalently to that in (B).
percentage of cells having ingested one to five spheres, or more than five spheres. By dividing the total number of macrophages having phagocytized one or more spheres by the number of cells within region 2 (Fig. 1A--macrophages), the percentage of the cells that phagocytized the particles can be determined. This can be further expanded into the percentage of phagocytic alveolar macrophages containing one, two, three, four, five, and more than five spheres by dividing the total number of phagocytic cells into the number containing one, two, etc., spheres. Sorted cells containing one to five microspheres are shown in Fig. 1C-G.
190
SPECIAL METHODS FOR MEASURING PHAGOCYTOSIS
[4]
Figure 2A-C illustrates the flow cytometric approach we recently used 8 to quantitate the migration of 1.9-/xm-diameter fluorescent (green) microspheres to the thoracic lymph nodes after 4 x 108 of these particles were deposited in the lungs of rats. Cells and particles liberated from the nodes after dissection were prepared in suspension and they were excited by the 488-nm line of an argon laser. Log 2° light scattering and green fluorescence over a 515- to 545-nm wavelength range were measured. Signal processing electronics were set to trigger on any incoming event, i.e., no coincidence requirements. Data were acquired for 30,000 events per sample and stored in the correlated list mode fashion.11 In order to estimate the total number of particles associated with the nodal cells in each analyzed sample, the integrated sum of fluorescent events coincident with region II of the log 2° light scattering histogram, as illustrated in Fig. 2A and C, was determined. Those particulate events falling in the window range for ->4 particles were assigned a minimum value of 4 for computational purposes. This sum was expressed as a fraction of the total number of cells analyzed in region II. This fraction, in conjunction with the total cell yield from the nodes of a given animal, was then used to determine the total numbers of particles associated with all the cells harvested. As with the alveolar macrophages, the particulate burdens in each nodal phagocyte were also determined. The total number of free or non-cell-associated particles in the nodal samples was determined by analyzing region I of the log 2 ° light scatter histogram, Fig. 2A, for the fluorescent particulate events shown in Fig. 2B. The total number of fluorescent particles coincident with region I were expressed as a fraction of the total number of cellular events occurring in region II, and this fraction, in turn, was used in conjunction with nodal cell yields to determine the total number of "free" particles harvested from the lymph nodes. Comments
We have described experimental techniques whereby phagocytosis by mononuclear phagocytes can be rapidly and accurately measured using multiparameter flow cytometry. Quantitative indices of phagocytosis obtained using the flow cytometric approach include the percentage of cells that actually phagocytose particles, and the distribution of particles in the cells that contain them. Therefore, the relative number of cells in a population that has performed the work of phagocytosis, as well as an index of the extent of the work performed by the phagocytic cells, is precisely obtained. Moreover, the in vitro suspension assay system outlined here
[4]
FLOW CYTOMETRY AND PHAGOCYTOSIS
191
allows one to obtain these quantitative indices of phagocytosis by evaluating the phagocytic activity of virtually every cell in an experimental population, unlike some conventional phagocytic assay systems that use monolayers of mononuclear phagocytes formed with adherent subpopulations only. As well, performance of the phagocytic assay with cells and particles in a well-mixed system negates the possibility that the phagocytic activities of the cells may be altered by adherence to a stationary substrate. 12 Furthermore, removal of adherent cells from a substrate is accompanied by death of a large proportion of the cells. The phagocytic assay presented in this chapter, by the nature of the test particles used, measures "nonspecific" phagocytosis. However, since the fluorescent microspheres can be obtained with carboxyl groups, it may be possible to suitably coat them with appropriate opsonins for studying specific receptor-mediated phagocytosis. The finding by van Oss and co-workers that IgG adsorption onto polystyrene spheres augments their phagocytosis by granulocytesl3 lends tentative support for this possibility. Other investigators have used fluorescently labeled biotic particles such as bacteria in conjunction with flow cytometry to measure specific receptor-mediated phagocytosis. 14,15The high coefficient of variation in the fluorescence from these biological test particles, however, restricts their usefulness. The percentage of cells that phagocytize can be determined, but individual cell burdens of particles cannot presently be resolved. Regardless, the microsphere assay that employs unopsonized particles may be especially appropriate for measuring the phagocytic activities of cells like the alveolar macrophages, inasmuch as these phagocytes routinely encounter particles deposited in the alveolar microenvironment, which is relatively deficient in proteins and opsonins.16 In summary, we know of no other automated method, except for the flow cytometric technique described here, that can measure the percentage of a macrophage population that has phagocytized, and also gives discrete information on particulate burdens in individual cells. The flow cytometric approach offers major advantages over existing, conventional techniques. Chiefly, data can be easily obtained from large sample sizes and, because of the rapidity of the method, multiple cell samples can be evaluated over a short period of time. 12 B. E. Lehnert and P. E. Morrow, Immunol. Commun. 13, 313 (1984). t3 C. J. van Oss and M. W. Stenson, RES: J. Reticuloendothel. Soc. 8, 397 (1970). 14 p. Szejda, J. W. Parce, M. S. Seeds, and D. A. Boss, J. Immunol. 133, 3303 (1984). 15 C. F. Bassoe, O. D. Laerum, J. Glette, G. Hopen, B. Haneberg, and C. O. Solberg, Cytometry 4, 254 (1983). 16 H. Y. Reynolds and H. N. Newhall, J. Lab. Clin. Med. 84, 559 (1974).
192
SPECIAL METHODS
FOR MEASURING
PHAGOCYTOSIS
[5]
Acknowledgments This work was supported in part by the Department of Energy, Office of Health and Environmental Research, and the Los Alamos National Flow Cytometry and Sorting Research Resource funded by the Division of Research Resources of NIH (Grant No. P41-RK 01315-02).
[5] O i l - D r o p l e t M e t h o d for M e a s u r i n g P h a g o c y t o s i s
By THOMAS P. STOSSEL Introduction The basis of the oil-droplet method to assay phagocytosis is to stabilize particles containing a marker dye or radioactive compound, feed them to phagocytes, separate uningested particles from the cells by differential centrifugation, extract the marker from the cells, and measure it. The rate of association of the marker with the centrifuged cell pellet as a function of time reflects the rate of phagocytosis. Since its initial description ~the oil-droplet method has been modified in many ways. Originally, the dye oil red O was incorporated as a marker into heavy paraffin oil. Subsequently, diisodecyl phthalate was found to have a more suitable density than paraffin oil. Other modifications have included the use of radioactive lipids as markers 2 and of spin-labeled cholestanone, which can be quenched by ascorbic acid, permitting assessment of the extent to which particles are either adherent to the cells or are incorporated within unsealed vacuoles. Extracellular particles or particles in unsealed vacuoles are quenched whereas particles within closed vacuoles a r e n o t . 3,4 Double-label experiments have been performed by feeding cells particles labeled with either oil red O or perylene, measuring the former spectrophotometrically and the latter fluorimetrically.5 The method has several advantages. The most important one is the fact that the density of suitable oil droplets is such that the efficiency of separation of uningested particles from phagocytes is very high; the ability to remove adherent particles markedly facilitates the precise quantitation of phagocytic rates. By varying the material utilized to stabilize the particles, the recognition mechanisms of phagocytosis can be studied. J T. P. Stossel, R. J. Mason, J. H. Hartwig, and M. Vaughan, J. Clin. Invest. 51, 615 (1972). 2 A. Forsgren, D. Schmeling, and D. Zettervall, Immunology 32, 491 (1977). 3 M. J. Ueda, T. Ito, S.-I. Ohnishi, and T. S. Okada, J. Cell Sci. 51, 173 (1981). 4 T. Ito, M. J. Ueda, T. S. Okada, and S.-I. Ohnishi, J. Cell Sci. 51, 189 (1981). 5 R. D. Berlin, J. P. Fera, and J. R. Pfeiffer, J. Clin. Invest. 63, 1137 (1979).
METHODS IN ENZYMOLOGY, VOL. 132
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.