Clin. exp. Immunol. (1979) 35, 155-157.
A method for the ultrastructural examination of cell monolayers cultured in plastic microtitre plates P. L. P E N F O L D Department of Immunology, Middlesex Hospital Medical School, London
(Received 4 August 1978)
SUMMARY
A method is presented by which cells growing as monolayers cultured in microtitre plates can be embedded for electron microscopy. The technique has the following advantages: numerous specimens may be prepared with relatively small numbers of cells, cell-cell interactions remain undisturbed and may be enumerated, and morphology can be studied under circumstances identical with those used in cell-mediated cytotoxicity assays.
INTRODUCTION Examination of cell monolayers by electron microscopy frequently involves either embedding on glass slides or coverslips, or removal of cells with a rubber scraper. Embedding on glass is difficult, however, since splinters often remain and hinder section cutting; moreover, modern culture techniques do not utilize glass vessels. Removing the cultured cells by scraping without disturbing the monolayers is impractical. Although methods for embedding on plastic surfaces have been published recently (Perre & Foncin, 1977), no methods have been described specifically for microtitre plate cultures, a system frequently used by cellular immunologists. This technique was developed initially for the ultrastructural examination of interactions occurring between tumour target cells and immune spleen cells, in conjunction with an assay for cell-mediated cytotoxicity (Penfold & Jones, 1978). Cytolytic activity was measured by analysis of 5 'Cr release (Brunner et al., 1968) from mixtures of effector and target cells in flat bottomed microtitre plates (Sterilin, Middlesex). Interactions between human peripheral blood lymphocytes and antibody-coated tumour cells (Shen et al., 1978) have also been studied by this method. In order to avoid disturbing the cellular interactions so that the same relationships which existed in the assay system could be visualized in the electron microscope, cells were fixed and embedded in situ. METHODS AND RESULTS Before fixation the monolayers are vulnerable to disturbances, so care must be taken when adding the fixative to prevent lightly attached cells from being displaced and washed away. Fixatives are prepared at twice the required final strength and added very slowly by Finn pipette to an equal quantity of medium present in the wells. After fixation with glutaraldehyde, cells adhere more firmly to the plastic so that subsequent processing is less difficult. For routine morphological examination, 400 glutaraldehyde in 0-2 M cacodylate buffer pH 7 4, and 10 OS04 as post-fixative are prepared. Other fixatives may be used for histochemical analyses. Regimes which reveal ATPase activity (Penfold & Jones, 1978) and peroxidase activity (Graham & Karnovsky, 1966) have been applied. There was good penetration of fixatives and enzyme substrates with these procedures. Correspondence: Dr P. L. Penfold, Department of Immunology, Middlesex Hospital Medical School, Arthur Stanley House, 40-50 Tottenham Street, London WIP 9PG. 0099-9104/79/0010-0155$02.00 (0 1979 Blackwell Scientific Publications
155
156
P. L. Penfold
FIG. 1. P815Y (H-2d) tumour cells and C57BI (H-2") effector cells were cultured in microtitre plates at concentrations of 2 x 10' and 1F3 x l0' cells per well, respectively. The photograph shows a tumour cell, a junction-forming lymphocyte (T cell), a neutrophil and unattached lymphocytes, which have remained on the base of the microtitre plate. (Original magnification x 5000.)
Specimens are dehydrated in graded ethanol. Epoxypropane is not used since it dissolves the plastic (as does acetone) and therefore the final stage of dehydration is carried out with a mixture of equal parts of araldite and ethanol. Two to four drops of the 50/50 mixture are put into each well and left overnight that the ethanol evaporates, after which the wells are filled with fresh araldite. For best results the resin should be cured below 50'C for 2-3 days. Higher curing temperatures tend to produce distortions in the base of the micro-wells. After curing, the microtitre plates are cut into small sections with a fine-bladed saw. The aralditefilled wells are plunged into liquid nitrogen, so that differential contraction fractures the plastic and allows the araldite-embedded monolayers to be removed for sectioning. Electron micrographs of cell cultures embedded by this method are shown in Figs 1 and 2. so
DISCUSSION The method for the fixation and embedding of cell monolayers in situ described here makes it possible to study and enumerate cellular interactions under precisely the same circumstances used for in vitro biological assays. Additionally, artefacts resulting from special processing of cultures, such as the removal of cells from the plastic before fixation, are avoided. Furthermore, subtle interactions between cells are likely to be preserved. Since this method does not involve modification of existing culture conditions, it is readily applicable to a number of different in vitro systems. The method of Perre & Foncin (1977) was not directly applicable to microtitre plate cultures since, in our experience, the conditions recommended for curing the resin lead to distortions in the base of the micro-wells, although this is less pronounced with confluent monolayers. Embedding and sectioning in microtitre plates circumvents
Embedding monolayers
157
FIG. 2. SL2 tumour cells (a murine lymphoma) and human peripheral blood T cells were cultured in micro. titre plates at concentrations of 5 x 105 and 2 x 10' cells per well, respectively (Shen et al., 1978). The morphological features and granule content of the lymphocyte shown make it classifiable as a 'T gamma cell' (TG) (Grossi et al., 1978). It is associated with the remains of a lysed target cell. (Original magnification x 7500.)
the problems associated with processing glass cultures monolayers, facilitates the examination of numerous experimental parameters and avoids the need to use large numbers of cells. This work was supported by a grant from the Medical Research Council. I thank Professor I. M. Roitt for helpful discussion of the manuscript, and Philippa Peck for the typing. I am grateful to Drs B. Jones and L. Shen for providing the cultured monolayers. REFERENCES BRUNNER, K.T., MAUEL, J., CEROTTINI, J.C. & CHAPUIs, B. (1968) Quantitative assay of the lytic action of immune
lymphoid cells on 51Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology, 14, 181. GRAHAM, R.C. JR. & KARNovsKY, M.J. (1966) The early stages of absorption of injected horseradish peroxidase into the proximal tubules of mouse kidney. J. Histochem. Cytochem. 14, 291. GROSSI, C.E., WEBB, S.R., ZIccA, A., LfDYARD, P.M., MORETTA, L., MINGARI, C. & COOPER, M.D. (1978) Morphological and histochemical analyses of two human T cell subpopulations bearing receptors for IgM or IgG. J. exp. Med. 145, 1405.
PENFOLD, P. & JoNES, B. (1978) Allograft cytotoxicity: differences between lytic and non-lytic junctions as revealed by ultrastructural histochemistry. Immunology, (In press.) PERME, J. & FoNcIN, J.-F. (1977) An embedding method for monolayer cell cultures for light and electron microscopy. Stain Technology, 52, 240. SaNI, L., LrDYARD, P.M., PENFOLD, P. & RoITT, I.M. (1978) Evidence for antibody-dependent cell-mediated cytotoxicity by T cells bearing receptors for IgG. Clin. exp. Immunol. (In press.)
A method for the ultrastructural examination of cell monolayers cultured in plastic microtitre plates P. L. P E N F O L D Department of Immunology, Middlesex Hospital Medical School, London
(Received 4 August 1978)
SUMMARY
A method is presented by which cells growing as monolayers cultured in microtitre plates can be embedded for electron microscopy. The technique has the following advantages: numerous specimens may be prepared with relatively small numbers of cells, cell-cell interactions remain undisturbed and may be enumerated, and morphology can be studied under circumstances identical with those used in cell-mediated cytotoxicity assays.
INTRODUCTION Examination of cell monolayers by electron microscopy frequently involves either embedding on glass slides or coverslips, or removal of cells with a rubber scraper. Embedding on glass is difficult, however, since splinters often remain and hinder section cutting; moreover, modern culture techniques do not utilize glass vessels. Removing the cultured cells by scraping without disturbing the monolayers is impractical. Although methods for embedding on plastic surfaces have been published recently (Perre & Foncin, 1977), no methods have been described specifically for microtitre plate cultures, a system frequently used by cellular immunologists. This technique was developed initially for the ultrastructural examination of interactions occurring between tumour target cells and immune spleen cells, in conjunction with an assay for cell-mediated cytotoxicity (Penfold & Jones, 1978). Cytolytic activity was measured by analysis of 5 'Cr release (Brunner et al., 1968) from mixtures of effector and target cells in flat bottomed microtitre plates (Sterilin, Middlesex). Interactions between human peripheral blood lymphocytes and antibody-coated tumour cells (Shen et al., 1978) have also been studied by this method. In order to avoid disturbing the cellular interactions so that the same relationships which existed in the assay system could be visualized in the electron microscope, cells were fixed and embedded in situ. METHODS AND RESULTS Before fixation the monolayers are vulnerable to disturbances, so care must be taken when adding the fixative to prevent lightly attached cells from being displaced and washed away. Fixatives are prepared at twice the required final strength and added very slowly by Finn pipette to an equal quantity of medium present in the wells. After fixation with glutaraldehyde, cells adhere more firmly to the plastic so that subsequent processing is less difficult. For routine morphological examination, 400 glutaraldehyde in 0-2 M cacodylate buffer pH 7 4, and 10 OS04 as post-fixative are prepared. Other fixatives may be used for histochemical analyses. Regimes which reveal ATPase activity (Penfold & Jones, 1978) and peroxidase activity (Graham & Karnovsky, 1966) have been applied. There was good penetration of fixatives and enzyme substrates with these procedures. Correspondence: Dr P. L. Penfold, Department of Immunology, Middlesex Hospital Medical School, Arthur Stanley House, 40-50 Tottenham Street, London WIP 9PG. 0099-9104/79/0010-0155$02.00 (0 1979 Blackwell Scientific Publications
155
156
P. L. Penfold
FIG. 1. P815Y (H-2d) tumour cells and C57BI (H-2") effector cells were cultured in microtitre plates at concentrations of 2 x 10' and 1F3 x l0' cells per well, respectively. The photograph shows a tumour cell, a junction-forming lymphocyte (T cell), a neutrophil and unattached lymphocytes, which have remained on the base of the microtitre plate. (Original magnification x 5000.)
Specimens are dehydrated in graded ethanol. Epoxypropane is not used since it dissolves the plastic (as does acetone) and therefore the final stage of dehydration is carried out with a mixture of equal parts of araldite and ethanol. Two to four drops of the 50/50 mixture are put into each well and left overnight that the ethanol evaporates, after which the wells are filled with fresh araldite. For best results the resin should be cured below 50'C for 2-3 days. Higher curing temperatures tend to produce distortions in the base of the micro-wells. After curing, the microtitre plates are cut into small sections with a fine-bladed saw. The aralditefilled wells are plunged into liquid nitrogen, so that differential contraction fractures the plastic and allows the araldite-embedded monolayers to be removed for sectioning. Electron micrographs of cell cultures embedded by this method are shown in Figs 1 and 2. so
DISCUSSION The method for the fixation and embedding of cell monolayers in situ described here makes it possible to study and enumerate cellular interactions under precisely the same circumstances used for in vitro biological assays. Additionally, artefacts resulting from special processing of cultures, such as the removal of cells from the plastic before fixation, are avoided. Furthermore, subtle interactions between cells are likely to be preserved. Since this method does not involve modification of existing culture conditions, it is readily applicable to a number of different in vitro systems. The method of Perre & Foncin (1977) was not directly applicable to microtitre plate cultures since, in our experience, the conditions recommended for curing the resin lead to distortions in the base of the micro-wells, although this is less pronounced with confluent monolayers. Embedding and sectioning in microtitre plates circumvents
Embedding monolayers
157
FIG. 2. SL2 tumour cells (a murine lymphoma) and human peripheral blood T cells were cultured in micro. titre plates at concentrations of 5 x 105 and 2 x 10' cells per well, respectively (Shen et al., 1978). The morphological features and granule content of the lymphocyte shown make it classifiable as a 'T gamma cell' (TG) (Grossi et al., 1978). It is associated with the remains of a lysed target cell. (Original magnification x 7500.)
the problems associated with processing glass cultures monolayers, facilitates the examination of numerous experimental parameters and avoids the need to use large numbers of cells. This work was supported by a grant from the Medical Research Council. I thank Professor I. M. Roitt for helpful discussion of the manuscript, and Philippa Peck for the typing. I am grateful to Drs B. Jones and L. Shen for providing the cultured monolayers. REFERENCES BRUNNER, K.T., MAUEL, J., CEROTTINI, J.C. & CHAPUIs, B. (1968) Quantitative assay of the lytic action of immune
lymphoid cells on 51Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology, 14, 181. GRAHAM, R.C. JR. & KARNovsKY, M.J. (1966) The early stages of absorption of injected horseradish peroxidase into the proximal tubules of mouse kidney. J. Histochem. Cytochem. 14, 291. GROSSI, C.E., WEBB, S.R., ZIccA, A., LfDYARD, P.M., MORETTA, L., MINGARI, C. & COOPER, M.D. (1978) Morphological and histochemical analyses of two human T cell subpopulations bearing receptors for IgM or IgG. J. exp. Med. 145, 1405.
PENFOLD, P. & JoNES, B. (1978) Allograft cytotoxicity: differences between lytic and non-lytic junctions as revealed by ultrastructural histochemistry. Immunology, (In press.) PERME, J. & FoNcIN, J.-F. (1977) An embedding method for monolayer cell cultures for light and electron microscopy. Stain Technology, 52, 240. SaNI, L., LrDYARD, P.M., PENFOLD, P. & RoITT, I.M. (1978) Evidence for antibody-dependent cell-mediated cytotoxicity by T cells bearing receptors for IgG. Clin. exp. Immunol. (In press.)
