EXPEHIhlENTAL AND MOLECULAR PATHOLOGY 23, 220-227 ( lg’if? )

A Plating Method for Preparation of Cells for Culture and

for Observation by Light or Electron Microscopy

EMMA SHELTON AXD JAN M. ORENSTEIN

Laboratory of Biochemistry, National Cancer Institute, Natiotml lnstittrtcs of flecrlth, Bethesda, IMaryland 2001 I

Received January 27, 1975

A plating technique that makes use of small volumes of cell suspensions ( l-10 ~1) is described. Its purpose is to permit microscopic examination or yro:Tagation in culture of the entire contents of the microdroplet. Cells are allowed to settle by gravity on Millipore filters where they rapidly and firmly attach after 20 minutes of incubation at 37°C. Such cells can be used for long- or short-term cultures and can be processed for light microscopy and transmission or scanning electron micros- copy. The method is particularly useful for preparing cell suspensions for scanning electron microscopy since it is possible to match light photomicrographs with scanning electron micrographs of identical cells.

A plating method has been devised that permits the observation of the morphology and interactions between individual cells that have settled by gravity upon the surface of Millipore filters. Living cells rapidly attach to the filters and after 15-20 minutes are so firmly bound that they remain in place throughout subsequent preparative procedures. Plated cells may be “cultured” for a few minutes to several hours in a moist incubator at 37°C. These short- term survival cultures permit one to monitor by morphological and chemical analyses such activities as, for example, phagocytosis or cellular secretion. Long- term cultures may be prepared by plating cells and allowing them to attach under sterile conditions and subsequently sealing them in diffusion chambers for ira viva propagation (Shelton and Rice, 1958).

The method has some advantages over previously described methods that use glass or plastic as a substrate for cell attachment (Boyde et al., 1972; Porter et al., 1972; Wetzel et al., 1973). It provides a way to identify a cell by light microscopy and then to view the same cell in the scanning or transmission electron micro- scope. The following description is explicit for mouse peritoneal cells, but with the exception of the method for obtaining the cells, the procedure applies to any cell suspension.

METHODS

A mouse is shaved on the ventral surface and killed by exsanguination under ether anesthesia or by CO2 intoxication induced by placing the animal on a screen elevated above a small amount of frozen CO2 in a closed container. Immediately after death, 0.5-l ml of fluid (phosphate buffered saline, tissue

Copyright 0 1975 by Academic Press, IIIC. All rights of reproduction in any form reserved

PLATING TECHNIQUE FOR SEM 231

culture fluid; see below) is injected intraperitoneally and the mouse rotated gently for 30 seconds to distribute the fluid over the viscera. The mouse is pinned on a board, the abdominal skin reflected and the fluid, withdrawn by Pasteur pipette through a small hole cut in the abdominal muscle, is placed in a plastic tube held in ice. If proper precautions are taken, the cell suspension is sterile. Aliquots of the cell suspension are appropriately diluted and counted (Cello- scope, Particle Data, Elmhurst, Ill.). Normal Strain A female mice, 126-196 days of age injected with 1 ml of fluid yield about 0.5 ml of fluid containing 4-6 x lo6 cells/ml ( Shelton et al., 1971)).

Preparation of Millipore Filters

Millipore filters (Millipore Filter Corp. Bedford, Mass. 01730) are porous structures composed of cellulose esters or similar polymeric materials that range in thickness from 0.25 to 150 PM depending on the Type. The standard filter contains a soluble, nonionic surfactant that is harmful to cells and must be removed. Thus, all filters are boiled for 5 minutes each in three changes of distilled water, dried on clean filter paper, and stored until used. We have plated cells on filters of many pore sizes, but have found the Type HA (0.45 PM pore size; 150 PM thick), Type TH (0.45 pM pore size; 25 ,uM thick), and Type VS (0.025 PM pore size; 150 $‘U thick) to be the most useful. Filters for use in diffusion chambers are sterilized by dry heat; 75°C for 48 hours.

Plating Fluid

The filters may be moistened with any fluid that is compatible with the health of the cell. Eagle’s minimum essential medium (MEM) buffered at pH 7.4 with 25 pM HEPES [4-( 2-hydroxyethyl ) -I-piperazinyl-ethane-2 sulfonic acid 1 buffer has been used successfully and Dulbecco’s phosphate buffered saline supple- mented with 10% homologous mouse serum (inactivated, 57°C for 30 minutes; absorbed at 37°C for 15 minutes with equal volume of washed sheep erythro- cytes) is also satisfactory. Bicardonate buffered fluids are not satisfactory because of poor control of pH.

Plating Procedure

Plating fluid, 0.40.6 ml, is pipetted into the middle of a 60-mm diameter Petri dish and an RA filter (45 mm diam; 1.2 PM pore size; 150 PM thick) is carefully placed on it so that the fluid spreads evenly and no bubbles are trapped beneath the filter. Small (6 mm diam) filters of a selected porosity are arranged on the RA filter and allowed to become saturated from below by equilibration for I5 minutes at 37°C in a humidified incubator. If the small filters are wetted from the top, capillary action will pull the cells over the edge and successful plating will not be achieved. The filters may be coded by punching small holes around the rim with a needle, but care must be taken to plate the droplet at a distance from the holes.

Plating is accomplished by allowing a droplet of cell suspension to form on the tip of a Lang-Levy constriction pipette and touching it to the middle of a small filter. One to ten microliters may be plated with ease. The droplet will form a circle l-5 mm in diameter within which all the cells are contained.

222 SHELTON AND ORENSTEIN

In order to prevent excessive flattening and cell rupture on HA or TH filters, care must be taken to ensure that sufficient fluid is maintained in the RA filter. The closed Petri dish is returned to the incubator where it is left for the desired time. Only unfixed cells can be satisfactorily plated because fixed cells do not adhere to the fi1ters.l

In Vitro and In Vivo Culture

Short-term survival cultures may be prepared without sterile precautions by incubating the plated cells for 6-8 hours on the RA filters. Alternatively, after the cells have been allowed to settle and attach for 20 minutes, the filters can be removed from the RA filter and submerged in 10 ml of tissue culture fluid in a 60-mm Petri dish for continued incubation. Cells may also be plated and incubated on filters that are floating on the surface of the culture fluid. If desired and after the cells have attached, these filters, too, may be pressed beneath the fluid surface for continued incubation.

For long-term cultivation of the cells in diffusion chambers, the cell suspension is kept sterile, plated under sterile conditions and the filters to which the cells have become bound, are enclosed in single diffusion chambers that are inserted by laparotomy into host mice (Shelton and Rice, 1958). The diffusion chambers are placed in a shallow bath of plating fluid in the bottom of a large Petri dish where the cells are kept moist until insertion into the host. Such plated cells remain viable for months.

Processing Filters for Microscopy

Light Microscopy. The small filters are carefully removed from the surface of the RA filter, inverted and immersed flat to the surface of the fixative in order to avoid the shearing effect of passing through the air-fluid interface at an angle. The cells are fixed in 2% glutaraldehyde buffered in 0.067 M sodium cacodylate, pH 7.4, containing 1% sucrose (buffer) for 30 minutes to 1 hour at room temperature and washed with mild agitation for 5 minutes each in three changes of buffer on the platform of a reciprocating shaker (Eberbach Corp., Ann Arbor, Michigan). At this point the filters can be stored in buffer or 70% ethanol or they may be processed further through histochemical procedures or a variety of stains. Toluidine blue has proved to be a useful general stain. If the cells are to be stained immediately, the clarity of the stain is enhanced by soaking the filters for 15 minutes in 70$ ethanol. They are brought to distilled water and slowly agitated in 0.01% toluidine blue buffered in 0.005 M sodium citrate pH 4.3, for 15-30 minutes. The filters are then individually passed through 7Os, 95%‘, 100% ethonal (two changes), 1 minute each with mild agitation, into 50%/507, ethanol/toluene, 2 minutes, and finally two changes of 100yO toluene, 2 minutes each before mounting in Permount (Fisher Scientific Co., Fairlawn, New J’ersey) under a glass cover slip on a microscope slide.

Transmission electron microscopy. Any standard fixation and embedding

1 In the study of exfoliative cytology in the clinical laboratory the Shl Millipore filter is used. With this technique also, it is emphasized that unfixed cells are essential for low ceil loss and preservation of cell morphology. (Application Report AR-24; Millipore Corp., Red- ford, Mass. 01730.)

PLATING TECHNIQUE FOR SEM 223

FIG. 1. Light (a) and scanning (b) images of the area of plated cells scored into quad- rants for orientation. Higher power light photomicrographs of individual cells within each quadrant are used for positive identification (see Fig. 8). Cells incubated for 20 minutes at 3’7°C. 160x.

schedule may be used, but staining and flat embedding allows one to follow specific cells, and, with special care, to section them. After fixation in buffered 2% glutaraldehyde and postfixation in 1% 0~0~ in buffer, the filters are placed in 0.5% uranyl acetate in 0.54% sucrose for 30 minutes, washed in O.SS% NaCl (saline), and stained with mild agitation in filtered 10% Harris’ hematoxylin in saline for 2 minutes. The filters are washed in saline and selected areas of cells are cut out under a dissecting microscope. The pieces of filter are dehy- drated through ethanol and propylene oxide and flat embedded in Epon. After embedding, the specific cells of interest can again be located and the block trimmed in such a way that they are included in the sections cut on the micro- tome.

Scanning electron microscopy. Cells may be examined and photographed first in the light microscope and then processed and photographed with the scanning electron microscope. Filters are processed and stained as for light microscopy except while in 70% ethanol the filter is scored with a fine needle to divide the area of cells into quadrants for orientation (Fig. la and lb). The filters are mounted in Permount under a cover glass that is raised above the microscope slide on thin glass shims so that the cells are protected from damage. The cells are photographed at various magnifications (Zeiss Photomicroscope: Kodak 2483 color or Panatomic-X black and white 35-mm film). A long-working-distance oil immersion objective, 63~) 1.25 N.A., provides sufficient resolution for positive identification of cell type and the position of the cell within the quadrant is noted for future orientation in the scanning microscope.

When light microscopy is completed, the covers slip and Permount are care- fully removed with lOO$ toluene. After returning the filter to SO? :50%) ethanol/ toluene and 100% ethanol, it is transferred through two changes, 5 minutes each, of 50%/5070 ethanol/chloroform and 30% ethanol/70% chloroform and then into three changes of dry chloroform. It is placed in a slot of an 8-chamber wire basket and dried by the critical point method by substitution with liquid COz in a SAMDRI PVT-3 (Tousimis Research Corp., Rockville, Ma.). The dry

224 SHELTON AND ORENSTEIN

FIG. 2. Mouse peritoneal cells plated on a TH filter and incubated 5 minutes at 37°C before fixation. After fixation the filter was incubated with 3,3’-diaminobenzidine tetrahydro- chloride to stain the polymorphonuclear leukocyte granules (arrow). A mast cell with dark granules, small and large lymphocytes, and macrophages are also seen. Nuclear and cyto- plasmic detail is easily discerned. 890x.

PLATING TECHNIQUE FOR SEM 225

filters are attached directly to l-cm diameter aluminum stubs with double-sided tape and silver paint is applied at the edge of the filter to make contact with the metal of the stub. After being coated with carbon and gold-palladium while rotating on a SAMSPIN (Tousimis Corp., Rockville, Md. ), the cells are examined on an ETEC Autoscan scanning electron microscope at a tilt of 5-45” with an accelerating voltage of 20 kV and photographed on Polaroid P/N 55 film.

RESULTS AND DISCUSSION

The plating technique makes use of very small volumes of fluid (l-10 ~1)

and its purpose is to allow the investigator to examine or propagate in culture the entire cellular contents of the microdroplet. One application of the technique

has already been described (Shelton et al., 1970). It is a method for making a permanent record of any cell suspension and several filters Lepresenting different experimental conditions can be accommodated on a single microscope slide. The cells are gently flattened as the droplets sink into the HA or TH filters SO that

cell types after staining can be identified with a high degree of accuracy (Figs. 2 and 5). The wetted VS filter provides a smooth-surfaced support (most suitable for scanning electron microscopy, Fig. 4) that maintains the plated peritoneal cell suspension as a convex drop for at least 6 hours of incubation. Cells settling

on these filters rapidly attach in characteristic ways (Shelton and Orenstein, 1974) and after 20 minutes of incubation at 37°C will not be dislodged by

subsequent fixation and processing (Fig. 3). Since many of the cells such as lymphocytes and some macrophages remain rounded-up for hours, their positive identification sometimes requires that photographs be taken at different levels

through the cell (Fig. 6a, b) and for this purpose, photographs in color are many times more useful than those in black and white for identifying individual cell types.

In addition to being a means for the quantitative and qualitative study of cell suspensions, the plating technique can be used to advantage in studying, by

FIG. 3. Cells plated on a VS filter and incubated for 2 hours at 37°C. A dark mast cell (top), flattened macrophages, and rounded lymphocytes can be identified. 890X.

FIG. 4. Scanning electron micrograph of a mast cell (center) and small lymphocyte (right). Behind and between these two cells is a sheep erythrocyte. These cells were processed directly for scanning electron microscopy with no intervening staining step. Compare the surface morphology of these cells with those in Fig. 8c that were stained with toluidine blue. 4500X.

FIG. 5. Three peritoneal lymphocytes (part of a macrophage can be seen at right) from a mouse immunized with horseradish peroxidase (HRP) and incubated on an HA filter for 15 minutes at 37°C in the presence of soluble HRP-anti-HRP immune complexes. After fixation, the HRP was visualized with the diaminobenzidine method of Graham and Kamovsky ( 1966). Both intra- and extracellular antigen can be seen. Compare with Fig. 7. 1560X.

FIG. 6a, b. Photographs at two different levels of a spontaneous rosette of sheep erythrocytes around a lymphocyte of a normal mouse. Cells were mixed with sheep erythrocytes and plated on a VS filter; incubation, 2 hours at 37°C. The rounded-up macrophage on the left can be identified by its characteristic nucleus. 1020x.

FIG. 7. Transmission electron micrograph of two lymphocytes from a normal mouse. Cells were mixed with soluble HRP-anti-HRP immune complexes, plated on TH filters and incu- bated 30 minutes at 37°C. The immune complexes visualized by the method of Graham and Karnovsky ( 1966), are localized in cytoplasmic vacuoles of one lymphocyte (left) and can also be seen between the two lymphocytes. Compare with Fig. 5. 11,700~.

226 SHELTON AND ORENSTEIN

FIG Sa-c. Light micrographs (a) of two mast cells, a IllacrOphdge (arrow) and lymph+ cytes are easily matched with the image on the viewing screen of the scanning electron microscope (b), thus, cells at higher magnification (c) can be identified. The enlarged image (c) defines the surface of a mast cell and two lymphocytes. Cells plated on a VS filter and incubated for 20 minutes at 37°C. Compare with Fig. 4 and note that the staining step has not visibly affected the surface morphology of these cells. (a) 1000X; (b) 1000x; (c) 7200x.

PLATING TECHNIQUE FOR SEM 227

light microscopy, the “patching” and “capping” of Fc receptors (Taylor et al., 1971) on the plasma membrane of peritoneal lymphocytes exposed to horse- radish peroxidase-anti-horseradish peroxidase immune complexes (Fig. 5)) and the formation of sheep erythrocyte rosettes around normal and immune lympho- cytes (Figs. 6a, b).

Cells plated on Millipore filters can be fixed and sectioned for transmission microscopy (Fig. 7) and filters of porosity greater than 0.22 pM can be processed as easily as pieces of tissue. The type “V” filters, with porosities of 0.1 pM or less are manufactured in two layers that split apart in the propylene oxide and result in diaphanous membranes that require meticulous care in embedding. Glauert ( 1974), who has recently reviewed the use of a variety of substrates that can serve as supporting layers for cells to be embedded for transmission

microscopy, recommends toluene instead of propylene oxide for Millipore filters. We feel that toluene would be of value for the “V” filters only.

The plating technique is particularly useful for observation of cells in the

scanning electron microscope. The VS filter surface is smooth but porous and cells such as lymphocytes, which do not readily adhere to glass or plastic, attach rapidly and firmly without exogenous pressure in a manner characteristic for

each cell type and because of this natural surface morphology is more likely to be preserved. Staining the cells with toluidine blue or processing them through

the histochemical procedure for localizing peroxidase activity (Graham and Karnovsky, 1966) does not visibly alter the structure of the cell membrane

(compare Fig. 4 with Fig. 8~). Where identification of cell type is important, light photomicrographs provide the means for doing so (Figs. la, b; 8a, b, c). It can become routine to photograph plated cells in the light microscope and

have enlarged prints at hand while selecting areas or individual cells to record with the scanning electron microscope.

REFERENCES BOYDE, A., WEISS, R. A., and VESELY, P. ( 1972). Scanning electron microscopy of cells in

culture. Exp. Cell Res. 71, 313324. GLAUERT, A. M. ( 1974). Fixation, dehydration and embedding of biological specimens. In

“Practical Methods in Electronmicroscopy” (A. M. Glauert, Ed.), Vol. 3, pp. l-201. Elsevier. GRAHAM, R. C., and KARNOVSKY, M. J. (1966). The early stages of absorption of injected

horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastrnctnral cyto- chemistry by a new technique. J. Histochem. Cytochem. 14, 291-302.

PORTER, K. R., KELLY, D., and ANDREWS, P. M. ( 1972). The preparation of cultured cells and soft tissues for scanning electron microscopy. In “Proc. 5th Annual Stereoscan Colloquium,” pp. l-9. Kent Cambridge Scientific, Inc., Chicago.

SHELTON, E., DAVES, L., and HEMMER, R. ( 1970). Q uantitation of strain BALB/c mouse peritoneal cells. Science 168, 1232-1234.

SHELTON, E., and ORENSTEIN, J, M. ( 1974). Localized membrane changes in lymphocytes and macrophages interacting with each other and with antigen. 1974. In “Proceedings of the 8th International Congress on Electron Microscopy” (J. V. Sanders, and D. J. Good- child, Eds.), Vol. 2. pp. 244-245. Austr. Acad. Sci., Canberra, Australia.

SHELTON, E., and RICE, M. E. (1958). Studies on mouse lymphomas II. Behavior of three lymphomas in diffusion chambers in relation to their invasive capacity in the host. J. Nat. Cancer Inst. 21, 137-161.

TAYLOR, R. B., DUFFUS, P. H., RAFF, M. C., and DE PETFIIS, S. ( 1971). Redistribution and pinocytosis of lymphocyte surface immunoglobulin molecules induced by anti-immuno- globulin antibody. Nature New Biol. 233, 225-229.

WETZEL, B., ERICKSON, B. W., and LEWIS, W. R. ( 1973). The need for positive identification of leukocytes examined by SEM. SEM/IITRI/1973, 535-542.