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Identification of proliferating lymphocyte subpopulations by combined alkaline phosphatase anti-alkaline phosphatase (APAAP) staining and autoradiography
Journal oflmmunologicalMethods, 90 (1986) 283-285
283
Elsevier JIM03989
Short communication
Identification of proliferating lymphocyte subpopulations by combined alkaline phosphatase anti-alkaline phosphatase (APAAP) staining and autoradiography. H. Schneider and R. Wachner University of Freiburg, Children's Hospital, Mathildenstrasse 1, 7800 Freiburg i. Br., F.R.G. (Accepted 13 March 1986)
An improvement in the classification of proliferating ([3H]thymidine incorporating) lymphocyte subpopulations in mitogen- or antigen-stimulated microcultures is described. The binding of subset-specific monoclonal antibodies is detected by the alkaline phosphatase anti-alkaline phosphatase method (APAAP). There are two advantages compared to the peroxidase anti-peroxidase (PAP) method; (1) endogenous enzyme (peroxidase) activity exhibited by some cells causes no interference, and (2) the red alkaline phosphatase staining obtained with new fuchsin provides a far superior contrast to silver grains than conventional peroxidase staining. Key words: Alkaline phosphatase anti-alkaline phosphatase," Peroxidase anti-peroxidase staining," Autoradiography
Introduction
Recently we described a micromethod which allows subpopulation classification of proliferating ([3H]thymidine incorporating) cells previously stimulated in microcultures (Schneider et al., 1984). The cells are stained for surface markers using the immuno-peroxidase method and proliferating cells are identified by subsequent autoradiography. However, some cells contain endogenous peroxidase activity which may partially or completely obscure either the specific immuno-peroxidase labelling or the silver grains of autoradiographs. This problem can be overcome by using immunoalkaline phosphatase (Cordell et al., 1984) instead of immuno-peroxidase.
Methods and results
Isolated mononuclear cells were cultured in microtitre plates and stimulated with mitogen (PHA,
ConA, PWM) or antigen (purified CMV antigen). 14 h before determining proliferating cell subsets, 20 /d [3H]thymidine solution (50 /~Ci/ml) with a specific activity of 2 Ci/mmol were introduced (cultures with mitogen on day 4, those with antigen on day 6). The cells were transferred to polyL-lysine-coated multispot slides and treated as outlined in detail previously (Bross et al., 1978). The APAAP staining procedure for cell surface markers was carried out as follows: (1) Add monoclonal antibodies: Leul, Leu2a, Leu3a, Leull, Leu12, diluted 1:50 (20 min); (2) Add rabbit anti-mouse IgG, dilution 1:40 (20 min); (3) Add mouse alkaline phosphatase anti-alkaline phosphatase (APAAP), dilution 1:100 (20 min); (4) Add naphthol AS-TR (as coupling agent) and new fuchsin (as capture agent), both as alkaline phosphatase substrate (15 min). The intensity of the APAAP labelling reaction could be enhanced by repeating steps 2 and 3 up
0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
284
285 to three times. Both steps could be shortened to 15 min. F o r the P A P technique: (1) A d d monoclonal antibodies: Leul, Leu2a, Leu3a, L e u l l , Leul2, diluted 1:50 (20 min); (2) A d d rabbit anti-mouse IgG, dilution 1:40 (5 min); (3) A d d swine anti-rabbit, dilution 1:20 (5 min); (4) A d d P A P from rabbit, dilution 1:20 (10 min); (5) A d d 3-amino-9-ethylcarbazole as substrate (15 min). F o r detecting [3 H]thymidine incorporating cells, the dried slides were overlaid with K o d a k A R - 1 0 stripping film. After 5 - 7 days of exposure the slides were developed with K o d a k D - 1 9 / 3 developer. The slides were covered with 80% glycerol-1% glutaraldehyde solution. Evaluation was performed under a light microscope (400 and 1000 x magnification). Positive cells exhibit a vivid red ring on the cell m e m b r a n e (Fig. 2).
Fig. 1. Mononuclear cells from cord blood cultivated for 4 days in the presence of PHA. 14 h before harvesting [3H]thymidine was added. The paired illustrations were photographed in different focal planes in order to maximize the separate components (silver grains and enzyme product). Cells are stained with the monoclonal antibody Leu2a employing the PAP technique followed by autoradiography (× 970). Eleven cells show incorporation of [3H]thymidine (Fig. la), four cells are Leu2a positive (Fig. lb). Of the proliferating cells (containing silver grains) three are positive for the Leu2a marker (arrowhead). One of these cells shows a typical capping phenomenon, the cell adjacent to it is slightly disintegrated but is clearly Leu2a positive. Two ceils (without silver grains) on the upper left side gave a faint positive reaction with PAP staining but from our experience they have to be judged as Leu2a negative. One cell (heavily labelled with silver grains) shows endogenous peroxidase activity and only the cytoplasm is stained (arrow). Fig. 2. Cells from a similar microculture as in Fig. 1 but stained using the APAAP technique. Thirty cells contain silver grains (Fig. 2a), eleven cells are Leu2a positive (Fig. 2b). Of the proliferating cells nine are positive for the Leu2a marker (arrowhead). There are two ceils not containing silver grains which are Leu2a positive, one of them shows the capping phenomenon. With the APAAP staining technique the association of the Leu2a marker with proliferating cells (marked by autoradiographic silver grains) is much easier to establish.
Conclusion The combination of surface marker staining and autoradiography permits the assignment of proliferating cells to subpopulations without employing separation procedures. I m m u n o - p e r oxidase labelling has been used previously but has the disadvantage, that reliable differentiation between the o r a n g e / b r o w n substrate product and photographic silver grains is not always easy. This is especially true when cells exhibit endogenous peroxidase activity, which may be so strong that the cytoplasmic staining obscures the silver grains completely. Staining with A P A A P and new fuchsin as a capture agent causes an intensive red m e m b r a n e labelling which gives a much better contrast to the silver grains (autoradiography) than the orange colour of the PAP technique (Figs. 1 and 2). In addition, the intensity of the A P A A P labelling reaction can be enhanced by repeating the staining procedare several times without risk of higher backgrounds. Recently the use of bromodeoxyuridine (BrdU) as an alternative to [3H]thymidine incorporation for the identification of proliferating cells has been described. BrdU incorporating cells can be stained with a commercially available monoclonal anti-BrdU a n t i b o d y (Gratzner, 1982; Morstyn et al., 1983). This technique would be a simple way of replacing laborious autoradiogaphic methods. However, in our technique simultaneous staining for surface markers and cytoplasmic BrdU is incompatible: preservation of surface markers with glutaraldehyde fixation prevents antibody access to BrdU and the alcohol treatment of cells which is performed to allow antibody penetration also destroys surface markers.
References Bross, K.J., C.G. Pangalis, C.G. Staatz and K.G. Blume, 1978. Transplantation 25, 331. Cordell, J.L., B. Falini, W.N. Erber, A.K. Gosh, Z. Abdulaziz. S. MacDonald, K.A.F. Pulford, H. Stein and D.Y. Mason, 1984, J. Histochem. Cytochem. 32, 219. Gratzner, H.G., 1982, Science 218, 474. Morstyn, G., S.M. Hsu, T. Kinsella, H. Gratzner, A. Russo and J.B. Mitchell, 1983, J. Clin. Invest. 72, 1844. Schneider, H., A. Vogt and K.J. Bross, 1984, Immunol. Commun. 13, 553.
283
Elsevier JIM03989
Short communication
Identification of proliferating lymphocyte subpopulations by combined alkaline phosphatase anti-alkaline phosphatase (APAAP) staining and autoradiography. H. Schneider and R. Wachner University of Freiburg, Children's Hospital, Mathildenstrasse 1, 7800 Freiburg i. Br., F.R.G. (Accepted 13 March 1986)
An improvement in the classification of proliferating ([3H]thymidine incorporating) lymphocyte subpopulations in mitogen- or antigen-stimulated microcultures is described. The binding of subset-specific monoclonal antibodies is detected by the alkaline phosphatase anti-alkaline phosphatase method (APAAP). There are two advantages compared to the peroxidase anti-peroxidase (PAP) method; (1) endogenous enzyme (peroxidase) activity exhibited by some cells causes no interference, and (2) the red alkaline phosphatase staining obtained with new fuchsin provides a far superior contrast to silver grains than conventional peroxidase staining. Key words: Alkaline phosphatase anti-alkaline phosphatase," Peroxidase anti-peroxidase staining," Autoradiography
Introduction
Recently we described a micromethod which allows subpopulation classification of proliferating ([3H]thymidine incorporating) cells previously stimulated in microcultures (Schneider et al., 1984). The cells are stained for surface markers using the immuno-peroxidase method and proliferating cells are identified by subsequent autoradiography. However, some cells contain endogenous peroxidase activity which may partially or completely obscure either the specific immuno-peroxidase labelling or the silver grains of autoradiographs. This problem can be overcome by using immunoalkaline phosphatase (Cordell et al., 1984) instead of immuno-peroxidase.
Methods and results
Isolated mononuclear cells were cultured in microtitre plates and stimulated with mitogen (PHA,
ConA, PWM) or antigen (purified CMV antigen). 14 h before determining proliferating cell subsets, 20 /d [3H]thymidine solution (50 /~Ci/ml) with a specific activity of 2 Ci/mmol were introduced (cultures with mitogen on day 4, those with antigen on day 6). The cells were transferred to polyL-lysine-coated multispot slides and treated as outlined in detail previously (Bross et al., 1978). The APAAP staining procedure for cell surface markers was carried out as follows: (1) Add monoclonal antibodies: Leul, Leu2a, Leu3a, Leull, Leu12, diluted 1:50 (20 min); (2) Add rabbit anti-mouse IgG, dilution 1:40 (20 min); (3) Add mouse alkaline phosphatase anti-alkaline phosphatase (APAAP), dilution 1:100 (20 min); (4) Add naphthol AS-TR (as coupling agent) and new fuchsin (as capture agent), both as alkaline phosphatase substrate (15 min). The intensity of the APAAP labelling reaction could be enhanced by repeating steps 2 and 3 up
0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
284
285 to three times. Both steps could be shortened to 15 min. F o r the P A P technique: (1) A d d monoclonal antibodies: Leul, Leu2a, Leu3a, L e u l l , Leul2, diluted 1:50 (20 min); (2) A d d rabbit anti-mouse IgG, dilution 1:40 (5 min); (3) A d d swine anti-rabbit, dilution 1:20 (5 min); (4) A d d P A P from rabbit, dilution 1:20 (10 min); (5) A d d 3-amino-9-ethylcarbazole as substrate (15 min). F o r detecting [3 H]thymidine incorporating cells, the dried slides were overlaid with K o d a k A R - 1 0 stripping film. After 5 - 7 days of exposure the slides were developed with K o d a k D - 1 9 / 3 developer. The slides were covered with 80% glycerol-1% glutaraldehyde solution. Evaluation was performed under a light microscope (400 and 1000 x magnification). Positive cells exhibit a vivid red ring on the cell m e m b r a n e (Fig. 2).
Fig. 1. Mononuclear cells from cord blood cultivated for 4 days in the presence of PHA. 14 h before harvesting [3H]thymidine was added. The paired illustrations were photographed in different focal planes in order to maximize the separate components (silver grains and enzyme product). Cells are stained with the monoclonal antibody Leu2a employing the PAP technique followed by autoradiography (× 970). Eleven cells show incorporation of [3H]thymidine (Fig. la), four cells are Leu2a positive (Fig. lb). Of the proliferating cells (containing silver grains) three are positive for the Leu2a marker (arrowhead). One of these cells shows a typical capping phenomenon, the cell adjacent to it is slightly disintegrated but is clearly Leu2a positive. Two ceils (without silver grains) on the upper left side gave a faint positive reaction with PAP staining but from our experience they have to be judged as Leu2a negative. One cell (heavily labelled with silver grains) shows endogenous peroxidase activity and only the cytoplasm is stained (arrow). Fig. 2. Cells from a similar microculture as in Fig. 1 but stained using the APAAP technique. Thirty cells contain silver grains (Fig. 2a), eleven cells are Leu2a positive (Fig. 2b). Of the proliferating cells nine are positive for the Leu2a marker (arrowhead). There are two ceils not containing silver grains which are Leu2a positive, one of them shows the capping phenomenon. With the APAAP staining technique the association of the Leu2a marker with proliferating cells (marked by autoradiographic silver grains) is much easier to establish.
Conclusion The combination of surface marker staining and autoradiography permits the assignment of proliferating cells to subpopulations without employing separation procedures. I m m u n o - p e r oxidase labelling has been used previously but has the disadvantage, that reliable differentiation between the o r a n g e / b r o w n substrate product and photographic silver grains is not always easy. This is especially true when cells exhibit endogenous peroxidase activity, which may be so strong that the cytoplasmic staining obscures the silver grains completely. Staining with A P A A P and new fuchsin as a capture agent causes an intensive red m e m b r a n e labelling which gives a much better contrast to the silver grains (autoradiography) than the orange colour of the PAP technique (Figs. 1 and 2). In addition, the intensity of the A P A A P labelling reaction can be enhanced by repeating the staining procedare several times without risk of higher backgrounds. Recently the use of bromodeoxyuridine (BrdU) as an alternative to [3H]thymidine incorporation for the identification of proliferating cells has been described. BrdU incorporating cells can be stained with a commercially available monoclonal anti-BrdU a n t i b o d y (Gratzner, 1982; Morstyn et al., 1983). This technique would be a simple way of replacing laborious autoradiogaphic methods. However, in our technique simultaneous staining for surface markers and cytoplasmic BrdU is incompatible: preservation of surface markers with glutaraldehyde fixation prevents antibody access to BrdU and the alcohol treatment of cells which is performed to allow antibody penetration also destroys surface markers.
References Bross, K.J., C.G. Pangalis, C.G. Staatz and K.G. Blume, 1978. Transplantation 25, 331. Cordell, J.L., B. Falini, W.N. Erber, A.K. Gosh, Z. Abdulaziz. S. MacDonald, K.A.F. Pulford, H. Stein and D.Y. Mason, 1984, J. Histochem. Cytochem. 32, 219. Gratzner, H.G., 1982, Science 218, 474. Morstyn, G., S.M. Hsu, T. Kinsella, H. Gratzner, A. Russo and J.B. Mitchell, 1983, J. Clin. Invest. 72, 1844. Schneider, H., A. Vogt and K.J. Bross, 1984, Immunol. Commun. 13, 553.