JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 14:298-306 (1990)

Scanning Electron Microscopic Study of Immunogold-Labeled Human Leukocytes ERNEST H. HELINSKI, GEORGE H. BOOTSMA, ROY J. McGROARTY, GERALDINE M. OVAK, ETIENNE DE HARVEN, AND JOHN L. PAULY Department of Molecular Immunology, Roswell Park Memorial Institute, Buffalo, New York (E.H.H., G.M.O., J.L.P.); Department of Pathology, University of Toronto, Toronto, Ontario, Canada M5G ZL5 (G.H.B., R.J.M., E.D.H.)

KEY WORDS

Colloidal gold, Critical point drying, Hairy cell leukemia, Interleukin-2 receptor, Macrophages, Lymphokine-activated killer cells, Peldri I1

ABSTRACT

For many years critical point drying (CPD) has been the method of choice for preparing cells for scanning electron microscopy (SEM). Described herein is a simple, efficient, inexpensive, reproducible, and safe procedure using Peldri 11, a proprietary fluorocarbon compound that is solid at room temperature and a liquid above 25”C, as a sublimation dehydrant for processing specimens for SEM. The utility of Peldri I1 was demonstrated in studies using leukocytes from the blood of healthy donors and patients with leukemia as well as from long-term lymphoblastoid cell lines. The application of the proposed Peldri TI procedure was further documented in SEM studies in which the expression and distribution of the interleukin-2 receptor (IL-2R) on leukocyte surface membranes was imaged using colloidal gold-labeled antibodies (i.e., immunogold). When compared with current SEM preparation procedures using CPD, Peldri I1 is a useful alternative that is thought to offer several important advantages.

INTRODUCTION The critical point drying (CPD) method, introduced for transmission electron microscopy, was reported several decades ago (Anderson, 1951). This method was revived with the advent of scanning electron microscopy (SEM) a t the end of the 1960s and has remained the major means of preparing biological specimens for SEM (reviewed in Bartlett and Burstyn, 1975; Cohen, 1974, 1977, 1979; Hayat and Zirkin, 1973). The popularity of CPD has been widely recognized, and different protocols have been prescribed to overcome certain drawbacks. Today, CPD remains the method of choice despite several limitations, (e.g., costly instrumentation, time-consuming multistep procedures, cell shrinkage, and potential hazards associated with highpressure chambers) [Cohen, 19791. We report here a simple, efficient, and safe sublimation dehydrant technique utilizing Peldri 11, a proprietary fluorocarbon compound that is solid a t room temperature and is a liquid above 25”C,as a n alternative to CPD for preparing human leukocytes for SEM.

MATERIALS AND METHODS Different human hematopoietic cells were used to define the utility of the Peldri I1 sublimation dehydration procedure described herein. These cells included freshly collected peripheral blood leukocytes of healthy adults and patients with leukemia as well a s lymphoblastoid cells of long-term normal or leukemic cell lines. To define the efficiency of our sublimation protocol, we focused our attention on the preservation of the surface morphology (e.g., microvilli, filopodia, and ruffles) of different human leukocyte subsets, particularly immunocompetent cells with immunogold-labeled cell surface receptors.

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Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy adults and patients with active “hairy cell” leukemia (HCL) using FicollPaquem and procedures recommended by the manufacturer (Pharmacia, Inc., Piscataway, NJ). Immunocompetent human macrophages were generated from PBMC and cultured as nonadherent cells in Teflon@ dishes (Helinski et al., 1988). Human leukemic T cells of the lymphoblastoid line MT-1 were used to define the application of the proposed sublimation procedure for processing immunogold-labeled cells. MT-1 cells are known to express constitutively the low-affinity subunit (55 kD) of the interleukin-2 receptor (IL-2R) (Lowenthal and Greene, 1987; Smith, 1988). Expression of the IL-2R by the MT-1 cells was also defined using the immunogold labeling procedure described below. The IL-2R was identified on normal human T cells that had been mitogen activated in short-term (e.g., 24 to 72 hr) cultures with purified phytohemagglutinin (PHA, 2.5pgiml; Burroughs Wellcome Co., Research Triangle Park, NC). Lymphokine (i.e., IL-2)-activated killer (LAK) cells were generated using purified human recombinant IL-2 (hrIL-2, 100 Unitsiml; kindly provided by the Cetus Corp., Emeryville, CA) and procedures that we have described previously (Mookerjee and Pauly, 1985). After 14 to 21 days of culture, the proliferating T cells (i.e., LAK-T cells) were harvested, and their antitumor cytolytic activity was assayed using human lung tumor cells of line A549 (ATCC CCL

Received February 21, 1989; accepted in revised form J u n e 20, 1989. Address reprint requests to Dr. John L. Pauly, Department of Molecular Immunology, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, NY 14263.

SEM STUDY OF IMMUNOGOLD-LABELED HUMAN LEUKOCYTES

185, American Type Culture Collection, Rockville, MD). The tumor cells were propagated on cover glasses that had been placed in the chambers of a 24-well plate. Conjugates of the killer cells and tumor cells were prepared by adding the LAK-T cells to culture wells of adherent tumor cells and cocultivating the two cell populations for 4 hr. Thereafter, the cover glasses were harvested, washed, fixed, and prepared for SEM examination using Peldri 11. The IL-2R receptor was identified by SEM using the following immunogold labeling procedure. Leukocytes to be examined (>95% viable; trypan blue dye-exclusion assay) were washed twice with Dulbecco’s calcium- and magnesium-free phosphate-buffered (pH 7.2) saline (PBS; Grand Island Biological Co., Grand Island, NY) by centrifugation (150g; 8 min, ambient temperature). The pelleted cells were resuspended in PBS to give a final cell density of 1 x 10s/ml. Then, 50 p1 of the viable cell suspension was distributed over the surface of a glass coverslip (12 mm diameter, No. 1 thickness; Fisher Scientific Co., Pittsburgh, PA). These coverslips, affixed to a 3 x 1 inch glass microscope slide with a rubber band, had been cleaned with chromic acid (Chromerge; Manostat, New York, NY) and coated with Alcian blue 8GX (Pauly et al., 1986) or freshly prepared poly-L-lysine (Mazia et al., 1975). The microscope slides were then placed in a stationary horizontal position in 37°C incubator having a humidified atmosphere of 10% COz and 95% air. At 10 to 15 min intervals, the adherence of the cells to the coated coverslips was monitored using a n inverted tissue culture microscope. After the cells had become attached (-30 m i d , the microscope slide was dipped several times into a Falcon 50 ml conical-shaped polypropylene centrifuge tube (Becton Dickinson Labware, Lincoln Park, NJ) containing PBS to remove nonadherent cells. The cells were then prefixed by placing the microscope slide for 10 min into a polypropylene Falcon centrifuge tube having a HEPES-buffered (0.2 M; pH 7.2) solution of 0.1% purified glutaraldehyde (EM grade; Polyscinces, Inc., Warrington, PA) and 4.5% sucrose in deionized water (412 mOsm). This prefix procedure was employed to prevent receptor endocytosis and loss of surface structure (de Harven, 1987; de Harven and Soligo, 1986). Thereafter, the slide was placed for 5 rnin in 0.1% glycine in PBS and then bathed twice for 5 rnin in PBS containing serum proteins to block nonspecific adsorption of the primary and/or secondary antibodies. This blocking solution (PBS-NHS-BSA) consisted of PBS with 1%heat-inactivated, filtered (0.22 pm) human serum (NHS) of a single healthy male donor (blood type AB/Rh+)and 1% bovine serum albumin (BSA; fraction V). The slide was removed and excess fluid was wiped off carefully. The expression of the IL-2R was defined using a double antibody (e.g., indirect) labeling procedure. For this purpose, the slide was placed in a horizontal position, and the monoclonal antibody (5 pg/ml PBS-NHS) was distributed over the surface of the cover glass containing the lymphocytes (anti-Leu-IL-BR, CD 25, p55, IgG1,; Becton Dickinson Immunocytometry Systems, Mountain View, CA). To define nonspecific antibody

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adsorption, other cells of the same preparation were exposed in a similar manner to a n isotypic control antibody (mouse myeloma, IgG1,, 5 pgiml PBS-NHS; Southern Biotechnology Association, Birmingham, AL). The slides were then returned to the humidified incubator for 1 hr. Unbound antibody was washed off by dipping the slide several times into a tube containing PBS and then allowing it to stand in clean PBS for 5 min. The thoroughly washed cells were then exposed to a secondary antibody that had been conjugated to colloidal gold (i.e., “immunogold”; reviewed by de Mey, 1983; de Waele et al., 1983). In this step, the cells that had been affixed to the cover glass and labeled with the primary antibody (monoclonal antibody or control antibody) were then exposed to purified goat antimouse IgG that had been conjugated to colloidal gold (e.g., usually 20 nm; Energy Beam Sciences, Agawam, MA). Specifically, the leukocytes were covered for 1h r (37°C) with 50 pl of gold-conjugated antibody that had been diluted 1:lO in PBS-NHS-BSA. In some experiments, the expression of the IL-2R and other membrane antigens was also defined by flow cytometry using leukocytes that had been labeled in a similar manner with the exception that the secondary antibody was a n FITC-fluorochrome-conjugated goat antimouse IgG (Zymed Laboratories, Inc., San Francisco, CA). Regardless of the leukocyte subset (e.g., T cells, B cells, NK cells, and macrophages), type (e.g., normal or leukemic), or source (e.g., freshly collected blood cells or cultured cells), the following Peldri I1 protocol proved to be a n efficient and reproducible means of processing the immunogold-labeled cells for SEM. After reacting the cells to the primary antibody and the gold-conjugated secondary antibody, the coverslip was then placed on the bottom of a 10 ml Pyrex beaker (with the cell-side of the cover glass facing up) having 8.0 ml of protein-free PBS so as to wash the cells thoroughly. After 15 min, 7.5 ml of the PBS was removed by aspiration and replaced with 7.5 ml of 2.5% glutaraldehyde in 0.1 M HEPES buffer (pH 7.2), and the beakers were then stored overnight at 4°C. Thereafter, the cells were washed (twice with PBS) and dehydrated (ethanol: 50%, 75%, 95% [twice], 100% [thrice]; 15 min each). When changing solutions, a small amount of fluid (-0.5 ml) was allowed to remain so as to prevent cell drying. Peldri 11 (Ted Pella, Inc., Redding, CAI was warmed to 45°C to liquify and then diluted with a n equal volume of absolute ethanol from a freshly opened bottle. (Note: Peldri I1 should be used under the protection of a chemical fume hood, and other safety precautions prescribed by the manufacturer should be exercised.) To each beaker containing a cover glass was then added 2.0 ml of the 50% Peldri I1 solution (ambient temperature). After 1hr, the beakers were placed on a microscope slide warmer (35°C; Fisher Scientific). USing a prewarmed glass pipette, the 50% Peldri I1 solution was removed and replaced quickly with 1.0 ml of 100% Peldri 11. After 30 min, cell infiltration with 100% Peldri I1 was repeated a second time. Sublimation of the Peldri I1 was performed in a

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E.H. HELINSKI ET AL

Fig. 1. Sublimation apparatus used for the controlled dehydration of specimens processed with Peldri

I1 for SEM. A detailed description of the equipment and its operation is presented in Materials and Methods.

chemical fume hood in an air-conditioned room having a temperature of less than 23.8% with the apparatus shown in Figure 1. This apparatus consisted of A: a n aspirator bottle (500 ml) filled with CaS04, indicating desiccant (W.A. Hammond Drierite Co., Xenia, OH); B: thumb-screw glass needle valve; C: vacuum desiccator (Nalge Co., Rochester, NY) with the stopcock in the open position (a half-inch hole was drilled into the dome, and a plastic tube connector for a rubber hose was cemented into position so as to provide a n exhaust); D: vacuum gauge (VWR Scientific, San Francisco, CA); E: a Styrofoam box, the front panel of which was removed for this illustration (in Fig. l ) , containing an 8 inch diameter coil of a 4 ft x 0.50 inch copper tubing that is marketed for refrigeration and household plumbing (Cerro Copper Products, Inc., Chicago, IL); the box was filled with crushed dry ice and sealed with a Styrofoam lid [(not illustrated in Fig. 11); F: gas purifier column containing activated charcoal, lower half, and desiccant, upper half; and G: vacuum pump (VWR Scientific).

Beakers containing coverslips with cells that had been permeated with 100% Peldri I1 were placed in the prechilled vacuum desiccator. With the thumb screw in the closed position, the pump was adjusted to provide a maximum vacuum (gauge reading, -60 cm Hg). Then the thumb screw was opened partially to reduce the guage reading by about one-third (-40 cm Hg) so as to provide a steady flow of dry air through the desiccator. Sublimation of the Peldri I1 under vacuum could be viewed through the clear dome of the desicator; for a run of six to eight beakers, sublimation was usually completed within 1.0 to 1.5 hr. The beakers were then placed in a 37°C oven for 15 min; thereafter, the coverslips were mounted onto aluminum SEM stubs using a silver conducting paint (Ernest F. Fullam, Inc., Latham, NY). Coverslips having cells that were not labeled with immunogold were then sputter coated with gold (thickness, -200 A; vacuum, -1 x lop1 Torr) using a model E5100 SEM Coating Unit (Polaron Instruments, Inc., Hatfield, PA). Coverslips with cells that had been labeled with immunogold

SEM STUDY OF IMMUNOGOLD-LABELED HUMAN LEUKOCYTES

Fig. 2. Morphology of human monocyte-derived macrophages that had been cultivated for 11 days before harvesting and processing for SEM using Peldri 11. a: Low magnification of large, adherent macrophages and small, spherical lymphocytes. x 1,500. b: Macrophages displaying typical ruff led membrane architecture. Note the filopodia that anchor the phagocytic cell to the substrate. x 7,000. c: Pleomor-

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phic nature of the surface membrane of macrophages is illustrated by this cell that displays numerous microvilli and a membrane architecture significantly different than the macrophages shown in the previous panel. x 6,000. d High magnification of the fillipodia illustrating the preservation of these delicate membrane structures obtained using the proposed Peldri I1 procedure. x 9,000,

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Fig. 3. Scanning electron micrographs of peripheral blood mononuclear cells isolated from a 65-year-old male with hairy cell leukemia (WBC, 60,500/mm3).Preservation of the membrane ultrastructure, particularly the retention of the hairy cell microvilli that are characteristic of this leukemia documents the utility of the proposed Peldri I1 procedure. a: Low-power magnification of leukocytes isolated

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from the blood using Ficoll-Paque. x 200. b Uniform morphology of this clonal population of leukemic cells. x 1,200, c: Seven cells displaying hairy microvilli that aid in establishing the diagnosis of this leukemia. x 4,000. d: High magnification of a representative cell illustrating the well-preserved morphology. x 11,000.

SEM STUDY OF IMMUNOGOLD-LABELED HUMAN LEUKOCYTES

Fig. 4. Expression of the IL-2R on the surface of HTLV-1-infected human leukemic T cells of the long-term lymphoblastoid cell line MT-1. Expression of the receptor for the T cell growth-promoting lymphokine IL-2 was defined using purified mouse monoclonal antibody anti-Leu-IL-2R, and this antibody was imaged using goat antimouse IgG that had been conjugated with colloidal gold (20 nm). Immunogold-labeled cells were processed for SEM using Peldri 11. a: Low

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magnification SEI mode of a single MT-1 leukemic T cell. x 6,500. b: Mixed SEIiBEI mode (normal polarity) of the area identified in a panel with a white box illustrating the expression and distribution of the IL-OR on the leukocyte surface membrane. x 25,000. c: BE1 mode (reversed polarity) of IL-2R sites present on the membrane and, specifically, the same area of the cell shown in a and b. x 25,000.

were coated with evaporated carbon (thickness, -200 RESULTS A; vacuum, -7.5 x lop6 Torr) using a model E306 Human monocyte-derived macrophages that had high-vacuum carbon coater (Edwards High Vacuum been cultivated as a nonadherent cell population in International, Sussex, England). petri dishes having a Teflon liner and that had been For electron microscopy examinations, gold-coated prepared for SEM using Peldri I1 are shown in Figure cells were usually examined using an ETEC Autoscan 2. These cells, harvested from 11-day-oldcultures, were SEM, and carbon-coated cells we viewed with a JEOL incubated on a glass coverslip that had been coated JSM 840 instrument equipped with a lanthanum with Alcian blue for 15 min before fixation. Figure 2a hexaboride (LaB,) cathode (accelerating potential, 20 illustrates the large size and distinct morphology of kV; probe current, 2 x 10-l' amps; working distance, these macrophages, which can readily be distinguished 8 mm). Secondary electron image (SEI)mode, backscat- from several lymphocytes that are present and that ter electron image (BEI) mode, and mixed image (SEI/ appear as small, spherical cells. The preservation of the BEI) mode were used to examine immunogold-labeled membrane ultrastructure, particularly filopodia that cells (de Harven et al., 1984; de Harven and Soligo, anchor the cells, is further illustrated in Figure 2b-d. 1986; de Harven, 1987). Peldri I1 has also proven useful in preparing freshly

SEM STUDY OF IMMUNOGOLD-LABELED HUMAN LEUKOCYTES

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Fig. 6. Close-up view of human killer lymphocytes and human tumor cells that illustrate the initial phase of the immunological reaction that involves tumor cell recognition and effector-to-target cell adhesion. Cytolytic LAK-T cells were prepared by cultivating normal human blood lymphocytes with hrIL-2 for 15 days. Human lung tumor cells were from line A549, and these adherent cells were propa-

gated on glass coverslips. a: Conjugate of a single LAK-T cell and human lung tumor cell that was prepared by cocultivating populations of the two cell types for 4 hr. x 15,000. b: Another view of the interaction of LAK-T cell with a human lung tumor cell. Note the absence of breaks in the fragile membrane structures of the LAK-T cell that binds this killer cell to the tumor target cell. X 15,000.

isolated blood leukocytes for SEM. This is illustrated in Figure 3, which presents low and high magnification views of blood leukocytes from a patient with hairy cell leukemia. Noteworthy is the well-preserved surface ultrastructure of these cells as well as the debris-free background. The proposed Peldri I1 procedure has been successfully employed for preparing immunogold-labeled cells for SEM. Shown in Figure 4 is a human leukemic T cell of the line MT-1, known to harbor the human T cell leukemia virus (HTLV-1). Greater than 90% of the cells labeled positively for IL-2R. A representative T cell is illustrated in Figure 4a. Numerous gold-labeled

IL-2R sites are identified, and their uniform distribution on the cell surface is illustrated in the mixed SEIi BE1 mode presented in Figure 4b. The white dots (BEI, normal polarity) present on this leukemic T cell are the 20 nm colloidal gold particles that had been conjugated to the secondary antibody. The number of labeled IL2R sites present on the surface of this cell is best demonstrated in the BE1 mode (Fig. 4c) in which the gold appears a s black particles (reverse polarity). The same immunogold labeling procedure was used successfully to demonstrate the rapid expression of the IL-2R on the surface of mitogen-activated normal T cells in short-term cultures. Very few (

Scanning electron microscopic study of immunogold-labeled human leukocytes.

For many years critical point drying (CPD) has been the method of choice for preparing cells for scanning electron microscopy (SEM). Described herein ...
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