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An improved method for elimination of mycoplasmas from cell cultures
Journal of Immunological Methods, 103 (1987) 185-188 Elsevier
185
JIM 04484
An improved method for elimination of mycoplasmas from cell cultures H. Kreipe, H.J. Radzun, A. Keulers and M.R. Parwaresch Institute of Pathology, University of Kiel, Hospitalstr. 42, 2300 Kiel, F.R.G. (Received 3 February 1987, revised received 28 April 1987, accepted 11 May 1987)
Cell lines infected by different species of mycoplasma (Mycoplasma orale, Mycoplasma hominis) were decontaminated by co-culture with human blood monocyte (BM)-derived macrophages and pooled human immunoglobulin preparations. Co-cultures with BM-derived macrophages or murine peritoneal macrophages (PM) alone were not successful. The phenotype of infected cell lines did not differ from that of uninfected cell fines as revealed by morphological, enzymecytochemical, and immunocytochemical analysis. Key words: Mycoplasma elimination; Macrophage; Pooled human immunoglobulin
Introduction
Mycoplasma contamination of human and animal cell lines is a common problem of in vitro tissue culture. Infection of cell cultures with mycoplasmas can drastically influence the growth and metabolism of mammalian ceils (Shin and Van Diggelen, 1978). Furthermore, functional assays can be influenced severely by this commonly undetected contamination as pointed out by many investigators (Jakway and Shevach, 1984, Proust et al., 1985; Ruuth et al., 1985;). Several techniques for the elimination of mycoplasmas have been developed (Marcus et al., 1980; Schimmelpfeng et al., 1980; Triglia and Burns, 1983). Some of them carry the risk of mutation and poor viability after decontamination or of induction of resistance to the antibiotics used (Schmidt and Erfle, 1984). In order to cirCorrespondence to: H.J. Radzun, Institute of Pathology, Hospitalstr. 42, 2300 Kiel, F.R.G. Abbreviations: BM, blood monocytes; PM, peritoneal macrophages; TK, thymidine kinase.
cumvent these problems we have established a method of decontamination that is based on the co-culture of the infected cells with human blood monocyte (BM)-derived macrophages as described "by Triglia and Burns (1983). Because macrophages alone did not prove to be sufficient for a constant decontamination, pooled human immunoglobulin was also used. This combined method ensured reliable and constant elimination of mycoplasmas. Moreover, the immunoreactivity of thymidine kinase-deficient (TK-) U-937 cells with a panel of monocyte/macrophage-specific monoclonal antibodies was not influenced following the use of this decontamination procedure.
Materials and methods
Cell lines U-937 T K - cells (Kreipe et al., 1985), mouse myeloma cell line X-63 Ag8.653, and human fibroblasts WI-38 were kept in RPMI 1640 medium (Serva, Heidelberg, F.R.G.) supplemented with 10% fetal calf serum (Boehringer, Mannheim,
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
186 F.R.G.), 4 mM glutamine (Seromed, Munich, F.R.G.), 100 IU ml-a penicillin (Hoechst, Frankfurt, F.R.G.), 100 /~g m1-1 streptomycin (Heye, Berlin, Germany), and 20 mM Hepes buffer (Merck, Darmstadt, F.R.G.).
Detection of mycoplasmas Two methods for the detection of mycoplasmas were used. Firstly, the Hoechst dye 33258 (Hoechst) was used to stain extranuclear D N A as described by Hessling et al. (1980). 1 X 103 cells were checked for the diagnosis of successful liberation. Secondly, cell-free supernatants of treated and untreated cells were subjected to culture of mycoplasmas as described by Freundt (1983).
Elimination of mycoplasmas Mycoplasma-contaminated cells (Mycoplasma orale, Mycoplasma hominis ) were co-cultured with thioglycolate-elicited murine peritoneal macrophages (PM) as described by Schimmelpfeng et al. (1980). Briefly, PM were seeded at a density of 3.5 x 105 m1-1 in 24-well culture plates and allowed to adhere. After 24 h non-adherent cells were discarded and mycoplasma-infected cells were added in medium containing 100 #g m1-1 Lincomycine (Hoechst, Frankfurt, F.R.G.) and 100 /~g m1-1 Tylocine (Gibco, Washington, DC). Coculture lasted for 3 days followed by 3 days of culture in fresh medium and screening for myco-
plasmas. Alternatively cells were co-cultured with human BM separated as described by B r y u m (1968) and Bennett and Cohn (1966) and transformed into macrophages by prolonged in vitro culture over 14 days. Co-culture conditions and mycoplasma screening were essentially the same as described for murine PM (Triglia and Burns, 1983). In addition, 14-day-old human BM cultures were co-cultured with contaminated cells two times for 3 days in medium supplemented with 10% pooled human immunoglobulin (Gammavenin, Behringwerke, Marburg, F.R.G.) but lacking Lincomycine and Tylocine. After an additional 3 days of culture in fresh medium screening for mycoplasmas was performed and followed up over the next 4 weeks. In control experiments 14-day-old human BM or pooled human immunoglobulin alone were added to mycoplasma-infected cell lines two times for 3 days.
Phenotype analysis Cytospin preparations of cell lines before and after mycoplasma elimination were subjected to Pappenheim staining and staining for non-specific acid esterase (Mueller et al., 1975). U-937 T K cells were immunostained using a three-step immunohistochemical method (Stein et al., 1980) with monocyte/macrophage-specific monoclonal antibodies such as Ki-M1, Ki-M6, Ki-M7 (Kreipe et al., 1986), Ki-M8 (Radzun et al., 1987), O K M
TABLE I COMPARISON OF DIFFERENT PROCEDURES USED FOR MYCOPLASMA DECONTAMINATION Numbers correspond to experiments showing elimination of mycoplasmas by culture of supernatants (1) and by DNA staining (2)
Murine PM Human BM derived macrophages Human BM derived macrophages + pooled human IgG a NOt done.
U-937 TK- cell line Total (1) no. of cultures 9 5
3
X-63 AG8.653 cell line Total (1) no. of cultures n.d. a
(2)
n.d.
WI-38 cell line Total (1) no. of cultures n.d.
(2)
(2)
n.d.
6
2
1
2
0
0
3
0
0
6
6
6
2
2
2
3
3
3
187
(Breard et al., 1980), and anti-monocyte 1 and 2 (Monol and Mono2) (Ugolini et al., 1980).
Results
lines from the applied mycoplasma strains. Comparable decontamination was not achieved, when either BM-derived macrophages or immunoglobulin alone were applied two times over 3 days to mycoplasma-infected cell lines.
Results of mycoplasma elimination
Results of phenotypical analys&
When permanent cell lines contaminated with mycoplasmas were co-cultivated with murine PM complete elimination could not be achieved. As shown in Table I this procedure was successful in about 50% of the experiments performed. Comparable results were obtained when murine PM were replaced by human BM-derived macrophages (Table I). The viability and proliferation rate of all three cell lines tested were not influenced by this decontamination procedure. When mycoplasmainfected cell lines were co-cultured with human BM-derived macrophages and pooled human immunoglobulin a reduced growth rate was noted, that recovered when culture was continued in medium without immunoglobulin over 3 days. In six out of six experiments this procedure led to the decontamination of infected U-937 T K - cells (Table I). Mycoplasma-infected murine myeloma cell line X-63 Ag8.653 and human fibroblasts WI-38 were also freed of contamination by this procedure (Table I). Follow-up analysis over the next 4 weeks confirmed that the procedure had resulted in successful decontamination of the analyzed cell
On Pappenheim staining morphological changes were not observed in X-63 Ag8.653 myeloma cells, WI-38 fibroblasts, and U-937 T K - cells. In addition, mycoplasma-infected U-937 T K - cells did not show any changes in non-specific acid esterase content nor in antigen expression compared to uninfected U-937 T K - cells (Table II).
TABLE II IMMUNOCYTOCHEMICAL REACTIVITY OF MYCOPLASMA INFECTED AND MYCOPLASMA FREE U-937 TKCELLS W I T H M O N O C Y T E / M A C R O P H A G E SPECIFIC MONOCLONAL ANTIBODIES KiM1
KiM6
KiM7
KiM8
OKM
Monol
U-937 TKmycoplasmainfected ( + )
(+) +
-
+
-
U-937 TKmycoplasmafree
(+) +
-
+
-
(+)
Mono2
Discussion
Recently, we established a thymidine kinase-deficient mutant of the human histiocytic cell line U-937 (Sundstr~m and Nilsson, 1976) and showed that it functions as a fusion partner for the generation of macrophage hybrids (Kreipe et al., 1985). Because mycoplasmas can drastically diminish the yield of hybrids during HAT medium selection and because no information exists about the fusion properties of monocytes/macrophages, there is an urgent need for a reliable procedure to eliminate mycoplasmas and exclude their deleterious effects on fusion rates. We chose the decontamination procedure described by Schimmelpfeng et al. (1980) and Triglia and Burns (1983), because these methods do not carry the risk of poor viability and mutation as does the one introduced by Marcus et al. (1980) (see Schmidt and Erfle (1984)). Successful elimination of mycoplasmas in all cell fines and experiments (confirmed over 4 weeks following decontamination) could only be observed when 10% pooled human immunoglobulin was added to the co-culture with human BM-derived macrophages (Table I). The phenotype, viability, and growth behavior of the tested cell lines did not alter after decontamination with BM-derived macrophages and pooled human immunoglobulin. Furthermore, U-937 T K - cells showed a constant reactivity pattern with a panel of monocyte/macrophagespecific monoclonal antibodies during this decontamination procedure (Table II).
188 T h e a d d i t i o n of p o o l e d h u m a n i m m u n o g l o b u l i n c o u l d b e c o n s i d e r e d as a n i m p r o v e m e n t for e s t a b lished d e c o n t a m i n a t i o n p r o c e d u r e s of cell lines c o n t a i n i n g m y c o p l a s m a i n f e c t i o n s . It m a y also serve as a n a l t e r n a t i v e m e t h o d w h e n cell lines are i n f e c t e d b y m y c o p l a s m a s r e s i s t a n t to a n t i b i o t i c s . T h e e n h a n c e m e n t of m y c o p l a s m a k i l l i n g b y B M d e r i v e d m a c r o p h a g e s p r o b a b l y results f r o m a s t i m u l a t o r y effect of i m m u n o g l o b u l i n s o n B M p o s s i b l y t h r o u g h a n i n c r e a s e d release of lytic e n z y m e s ( F e r r e r i et al., 1986). A l t e r n a t i v e l y it m a y b e d u e to the i n c r e a s e d i m m u n o p h a g o c y t i c a b i l i t y of B M - d e r i v e d m a c r o p h a g e s .
References Bennett, W.E. and Cohn, Z.A. (1966) The isolation and selected properties of blood monocytes. J. Exp. Med. 123, 145. B~Syum,A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21 (suppl.), 77. Breard, J., Reinherz, E.L., Kung, P.C., Goldstein, G. and Schlossman, S.F. (1980) A monoclonal antibody reactive with human peripheral blood monocytes. J. Immunol. 124, 1984. Ferreri, N.R., Beck, L. and Spiegelberg, H.L. (1986) /3Glucuronidase release from human monocytes induced with aggregated immunoglobulins of different classes. Cell. Immunol. 98, 57. Freundt, E.A. (1983) Culture media for classic mycoplasma. Methods Mycoplasmol. 1, 127. Hessling, J.J., Miller, S.E. and Levy, N.L. (1980) A direct comparison of procedures for the detection of mycoplasma in tissue cultures. J. Immunol. Methods 38, 315. Jakway, J.P. and Shevach, E.M. (1984) Mycoplasma contamination: A hazard of screening hybridoma supernatants for inhibition of [3H]thymidine incorporation. J. Immunol. Methods 67, 337. Kreipe, H., Radzun, H.J., Karck, M. and Parwaresch, M.R. (1985) Establishment of thymidin kinase (E.C. 2.7.1.21)-deficient mutants of human monocytic cell line U-937. Cell. Immunol. 91, 498. Kreipe, H., Radzun, H.J. and Parwaresch, M.R. (1986) Phenotypic differentiation patterns of the human monocyte/macrophage system. Histochem. J. 18, 441.
Marcus, M., Lavi, V., Nattenberg, A., Rottem, S. and Markowitz, O. (1980) Selective killing of mycoplasmas from contaminated mammalian ceils in cell cultures. Nature 285, 660. Mueller, J., Brun del Re, G., Buerki, H., Keller, H.U., Hess, M.W. and Cottier, H. (1975) Nonspecific acid esterase activity: A criterion for differentiation of T and B lymphocytes in mouse lymph nodes. Eur. J. Immunol. 5, 270. Proust, J.J., Buchholz, M.A. and Nordin, A.A. (1985) A 'lymphokine-like' soluble product that induces proliferation and maturation of B-cells appears in the serum-free supernatant of a T cell hybridoma as a consequence of mycoplasmal contamination. J. Immunol. 134, 390. Radzun, H.J., Kreipe, H., B~Sdewadt, S., Barth, J., Hansmann, M.L. and Parwaresch, M.R. (1987) Ki-M8 monoclonal antibody reactive with an intracytoplasmic antigen of monocyte/macrophage lineage. Blood, in press. Ruutli, E., Ranby, M., Friedrich, B., Persson, F.H., Goustin, A., Leanderson, T., Coutinho, A. and Lundgren, E. (1985) Mycoplasma mimicry of lymphokine activity in T-cell lines. Scand. J. Immunol. 21,593. Schimmelpfeng, L., Langenberg, U. and Peters, J.H. (1980) Macrophages overcome mycoplasma infections of cells in vitro. Nature 285, 661. Schmidt, J. and Erfle, V. (1984) Elimination of mycoplasmas from cell cultures and establishment of mycoplasma-free cell lines. J. Exp. Cell Res. 152, 565. Shin, S.J. and Van Diggelen, O.P. (1978) Phenotypic alterations in mammalian cell lines after mycoplasma infection. In: G.J. McGarrity, D.G. Murphy and W.W. Nichols (Eds.), Mycoplasma infected cell cultures. Plenum Press, New York. Stein, H., Bonk, A., Tolksdorf, G., Lennert, K., Rodt, H. and Gerdes, J. (1980) Immune histological analysis of the organization of normal lymphoid tissue and non-Hodgkin's lymphomas. J. Histochem. Cytochem. 28, 746. SundstriSm, C. and Nilsson, K. (1976) Establishment and characterization of human histiocytic lymphoma cell line (U937). Int. J. Cancer 17, 565. Triglia, T. and Burns, G.F. (1983) A method for in vitro clearance of mycoplasma from human cell lines. J. Immunol. Methods 64, 133. Ugolini, V., Nunez, G., Smith, R.G., Stastny, P. and Capra, J.D. (1980) Initial characterization of monoclonal antibodies against human monocytes. Proe. Natl. Acad. Sci. U.S.A. 77, 6764.
185
JIM 04484
An improved method for elimination of mycoplasmas from cell cultures H. Kreipe, H.J. Radzun, A. Keulers and M.R. Parwaresch Institute of Pathology, University of Kiel, Hospitalstr. 42, 2300 Kiel, F.R.G. (Received 3 February 1987, revised received 28 April 1987, accepted 11 May 1987)
Cell lines infected by different species of mycoplasma (Mycoplasma orale, Mycoplasma hominis) were decontaminated by co-culture with human blood monocyte (BM)-derived macrophages and pooled human immunoglobulin preparations. Co-cultures with BM-derived macrophages or murine peritoneal macrophages (PM) alone were not successful. The phenotype of infected cell lines did not differ from that of uninfected cell fines as revealed by morphological, enzymecytochemical, and immunocytochemical analysis. Key words: Mycoplasma elimination; Macrophage; Pooled human immunoglobulin
Introduction
Mycoplasma contamination of human and animal cell lines is a common problem of in vitro tissue culture. Infection of cell cultures with mycoplasmas can drastically influence the growth and metabolism of mammalian ceils (Shin and Van Diggelen, 1978). Furthermore, functional assays can be influenced severely by this commonly undetected contamination as pointed out by many investigators (Jakway and Shevach, 1984, Proust et al., 1985; Ruuth et al., 1985;). Several techniques for the elimination of mycoplasmas have been developed (Marcus et al., 1980; Schimmelpfeng et al., 1980; Triglia and Burns, 1983). Some of them carry the risk of mutation and poor viability after decontamination or of induction of resistance to the antibiotics used (Schmidt and Erfle, 1984). In order to cirCorrespondence to: H.J. Radzun, Institute of Pathology, Hospitalstr. 42, 2300 Kiel, F.R.G. Abbreviations: BM, blood monocytes; PM, peritoneal macrophages; TK, thymidine kinase.
cumvent these problems we have established a method of decontamination that is based on the co-culture of the infected cells with human blood monocyte (BM)-derived macrophages as described "by Triglia and Burns (1983). Because macrophages alone did not prove to be sufficient for a constant decontamination, pooled human immunoglobulin was also used. This combined method ensured reliable and constant elimination of mycoplasmas. Moreover, the immunoreactivity of thymidine kinase-deficient (TK-) U-937 cells with a panel of monocyte/macrophage-specific monoclonal antibodies was not influenced following the use of this decontamination procedure.
Materials and methods
Cell lines U-937 T K - cells (Kreipe et al., 1985), mouse myeloma cell line X-63 Ag8.653, and human fibroblasts WI-38 were kept in RPMI 1640 medium (Serva, Heidelberg, F.R.G.) supplemented with 10% fetal calf serum (Boehringer, Mannheim,
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
186 F.R.G.), 4 mM glutamine (Seromed, Munich, F.R.G.), 100 IU ml-a penicillin (Hoechst, Frankfurt, F.R.G.), 100 /~g m1-1 streptomycin (Heye, Berlin, Germany), and 20 mM Hepes buffer (Merck, Darmstadt, F.R.G.).
Detection of mycoplasmas Two methods for the detection of mycoplasmas were used. Firstly, the Hoechst dye 33258 (Hoechst) was used to stain extranuclear D N A as described by Hessling et al. (1980). 1 X 103 cells were checked for the diagnosis of successful liberation. Secondly, cell-free supernatants of treated and untreated cells were subjected to culture of mycoplasmas as described by Freundt (1983).
Elimination of mycoplasmas Mycoplasma-contaminated cells (Mycoplasma orale, Mycoplasma hominis ) were co-cultured with thioglycolate-elicited murine peritoneal macrophages (PM) as described by Schimmelpfeng et al. (1980). Briefly, PM were seeded at a density of 3.5 x 105 m1-1 in 24-well culture plates and allowed to adhere. After 24 h non-adherent cells were discarded and mycoplasma-infected cells were added in medium containing 100 #g m1-1 Lincomycine (Hoechst, Frankfurt, F.R.G.) and 100 /~g m1-1 Tylocine (Gibco, Washington, DC). Coculture lasted for 3 days followed by 3 days of culture in fresh medium and screening for myco-
plasmas. Alternatively cells were co-cultured with human BM separated as described by B r y u m (1968) and Bennett and Cohn (1966) and transformed into macrophages by prolonged in vitro culture over 14 days. Co-culture conditions and mycoplasma screening were essentially the same as described for murine PM (Triglia and Burns, 1983). In addition, 14-day-old human BM cultures were co-cultured with contaminated cells two times for 3 days in medium supplemented with 10% pooled human immunoglobulin (Gammavenin, Behringwerke, Marburg, F.R.G.) but lacking Lincomycine and Tylocine. After an additional 3 days of culture in fresh medium screening for mycoplasmas was performed and followed up over the next 4 weeks. In control experiments 14-day-old human BM or pooled human immunoglobulin alone were added to mycoplasma-infected cell lines two times for 3 days.
Phenotype analysis Cytospin preparations of cell lines before and after mycoplasma elimination were subjected to Pappenheim staining and staining for non-specific acid esterase (Mueller et al., 1975). U-937 T K cells were immunostained using a three-step immunohistochemical method (Stein et al., 1980) with monocyte/macrophage-specific monoclonal antibodies such as Ki-M1, Ki-M6, Ki-M7 (Kreipe et al., 1986), Ki-M8 (Radzun et al., 1987), O K M
TABLE I COMPARISON OF DIFFERENT PROCEDURES USED FOR MYCOPLASMA DECONTAMINATION Numbers correspond to experiments showing elimination of mycoplasmas by culture of supernatants (1) and by DNA staining (2)
Murine PM Human BM derived macrophages Human BM derived macrophages + pooled human IgG a NOt done.
U-937 TK- cell line Total (1) no. of cultures 9 5
3
X-63 AG8.653 cell line Total (1) no. of cultures n.d. a
(2)
n.d.
WI-38 cell line Total (1) no. of cultures n.d.
(2)
(2)
n.d.
6
2
1
2
0
0
3
0
0
6
6
6
2
2
2
3
3
3
187
(Breard et al., 1980), and anti-monocyte 1 and 2 (Monol and Mono2) (Ugolini et al., 1980).
Results
lines from the applied mycoplasma strains. Comparable decontamination was not achieved, when either BM-derived macrophages or immunoglobulin alone were applied two times over 3 days to mycoplasma-infected cell lines.
Results of mycoplasma elimination
Results of phenotypical analys&
When permanent cell lines contaminated with mycoplasmas were co-cultivated with murine PM complete elimination could not be achieved. As shown in Table I this procedure was successful in about 50% of the experiments performed. Comparable results were obtained when murine PM were replaced by human BM-derived macrophages (Table I). The viability and proliferation rate of all three cell lines tested were not influenced by this decontamination procedure. When mycoplasmainfected cell lines were co-cultured with human BM-derived macrophages and pooled human immunoglobulin a reduced growth rate was noted, that recovered when culture was continued in medium without immunoglobulin over 3 days. In six out of six experiments this procedure led to the decontamination of infected U-937 T K - cells (Table I). Mycoplasma-infected murine myeloma cell line X-63 Ag8.653 and human fibroblasts WI-38 were also freed of contamination by this procedure (Table I). Follow-up analysis over the next 4 weeks confirmed that the procedure had resulted in successful decontamination of the analyzed cell
On Pappenheim staining morphological changes were not observed in X-63 Ag8.653 myeloma cells, WI-38 fibroblasts, and U-937 T K - cells. In addition, mycoplasma-infected U-937 T K - cells did not show any changes in non-specific acid esterase content nor in antigen expression compared to uninfected U-937 T K - cells (Table II).
TABLE II IMMUNOCYTOCHEMICAL REACTIVITY OF MYCOPLASMA INFECTED AND MYCOPLASMA FREE U-937 TKCELLS W I T H M O N O C Y T E / M A C R O P H A G E SPECIFIC MONOCLONAL ANTIBODIES KiM1
KiM6
KiM7
KiM8
OKM
Monol
U-937 TKmycoplasmainfected ( + )
(+) +
-
+
-
U-937 TKmycoplasmafree
(+) +
-
+
-
(+)
Mono2
Discussion
Recently, we established a thymidine kinase-deficient mutant of the human histiocytic cell line U-937 (Sundstr~m and Nilsson, 1976) and showed that it functions as a fusion partner for the generation of macrophage hybrids (Kreipe et al., 1985). Because mycoplasmas can drastically diminish the yield of hybrids during HAT medium selection and because no information exists about the fusion properties of monocytes/macrophages, there is an urgent need for a reliable procedure to eliminate mycoplasmas and exclude their deleterious effects on fusion rates. We chose the decontamination procedure described by Schimmelpfeng et al. (1980) and Triglia and Burns (1983), because these methods do not carry the risk of poor viability and mutation as does the one introduced by Marcus et al. (1980) (see Schmidt and Erfle (1984)). Successful elimination of mycoplasmas in all cell fines and experiments (confirmed over 4 weeks following decontamination) could only be observed when 10% pooled human immunoglobulin was added to the co-culture with human BM-derived macrophages (Table I). The phenotype, viability, and growth behavior of the tested cell lines did not alter after decontamination with BM-derived macrophages and pooled human immunoglobulin. Furthermore, U-937 T K - cells showed a constant reactivity pattern with a panel of monocyte/macrophagespecific monoclonal antibodies during this decontamination procedure (Table II).
188 T h e a d d i t i o n of p o o l e d h u m a n i m m u n o g l o b u l i n c o u l d b e c o n s i d e r e d as a n i m p r o v e m e n t for e s t a b lished d e c o n t a m i n a t i o n p r o c e d u r e s of cell lines c o n t a i n i n g m y c o p l a s m a i n f e c t i o n s . It m a y also serve as a n a l t e r n a t i v e m e t h o d w h e n cell lines are i n f e c t e d b y m y c o p l a s m a s r e s i s t a n t to a n t i b i o t i c s . T h e e n h a n c e m e n t of m y c o p l a s m a k i l l i n g b y B M d e r i v e d m a c r o p h a g e s p r o b a b l y results f r o m a s t i m u l a t o r y effect of i m m u n o g l o b u l i n s o n B M p o s s i b l y t h r o u g h a n i n c r e a s e d release of lytic e n z y m e s ( F e r r e r i et al., 1986). A l t e r n a t i v e l y it m a y b e d u e to the i n c r e a s e d i m m u n o p h a g o c y t i c a b i l i t y of B M - d e r i v e d m a c r o p h a g e s .
References Bennett, W.E. and Cohn, Z.A. (1966) The isolation and selected properties of blood monocytes. J. Exp. Med. 123, 145. B~Syum,A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21 (suppl.), 77. Breard, J., Reinherz, E.L., Kung, P.C., Goldstein, G. and Schlossman, S.F. (1980) A monoclonal antibody reactive with human peripheral blood monocytes. J. Immunol. 124, 1984. Ferreri, N.R., Beck, L. and Spiegelberg, H.L. (1986) /3Glucuronidase release from human monocytes induced with aggregated immunoglobulins of different classes. Cell. Immunol. 98, 57. Freundt, E.A. (1983) Culture media for classic mycoplasma. Methods Mycoplasmol. 1, 127. Hessling, J.J., Miller, S.E. and Levy, N.L. (1980) A direct comparison of procedures for the detection of mycoplasma in tissue cultures. J. Immunol. Methods 38, 315. Jakway, J.P. and Shevach, E.M. (1984) Mycoplasma contamination: A hazard of screening hybridoma supernatants for inhibition of [3H]thymidine incorporation. J. Immunol. Methods 67, 337. Kreipe, H., Radzun, H.J., Karck, M. and Parwaresch, M.R. (1985) Establishment of thymidin kinase (E.C. 2.7.1.21)-deficient mutants of human monocytic cell line U-937. Cell. Immunol. 91, 498. Kreipe, H., Radzun, H.J. and Parwaresch, M.R. (1986) Phenotypic differentiation patterns of the human monocyte/macrophage system. Histochem. J. 18, 441.
Marcus, M., Lavi, V., Nattenberg, A., Rottem, S. and Markowitz, O. (1980) Selective killing of mycoplasmas from contaminated mammalian ceils in cell cultures. Nature 285, 660. Mueller, J., Brun del Re, G., Buerki, H., Keller, H.U., Hess, M.W. and Cottier, H. (1975) Nonspecific acid esterase activity: A criterion for differentiation of T and B lymphocytes in mouse lymph nodes. Eur. J. Immunol. 5, 270. Proust, J.J., Buchholz, M.A. and Nordin, A.A. (1985) A 'lymphokine-like' soluble product that induces proliferation and maturation of B-cells appears in the serum-free supernatant of a T cell hybridoma as a consequence of mycoplasmal contamination. J. Immunol. 134, 390. Radzun, H.J., Kreipe, H., B~Sdewadt, S., Barth, J., Hansmann, M.L. and Parwaresch, M.R. (1987) Ki-M8 monoclonal antibody reactive with an intracytoplasmic antigen of monocyte/macrophage lineage. Blood, in press. Ruutli, E., Ranby, M., Friedrich, B., Persson, F.H., Goustin, A., Leanderson, T., Coutinho, A. and Lundgren, E. (1985) Mycoplasma mimicry of lymphokine activity in T-cell lines. Scand. J. Immunol. 21,593. Schimmelpfeng, L., Langenberg, U. and Peters, J.H. (1980) Macrophages overcome mycoplasma infections of cells in vitro. Nature 285, 661. Schmidt, J. and Erfle, V. (1984) Elimination of mycoplasmas from cell cultures and establishment of mycoplasma-free cell lines. J. Exp. Cell Res. 152, 565. Shin, S.J. and Van Diggelen, O.P. (1978) Phenotypic alterations in mammalian cell lines after mycoplasma infection. In: G.J. McGarrity, D.G. Murphy and W.W. Nichols (Eds.), Mycoplasma infected cell cultures. Plenum Press, New York. Stein, H., Bonk, A., Tolksdorf, G., Lennert, K., Rodt, H. and Gerdes, J. (1980) Immune histological analysis of the organization of normal lymphoid tissue and non-Hodgkin's lymphomas. J. Histochem. Cytochem. 28, 746. SundstriSm, C. and Nilsson, K. (1976) Establishment and characterization of human histiocytic lymphoma cell line (U937). Int. J. Cancer 17, 565. Triglia, T. and Burns, G.F. (1983) A method for in vitro clearance of mycoplasma from human cell lines. J. Immunol. Methods 64, 133. Ugolini, V., Nunez, G., Smith, R.G., Stastny, P. and Capra, J.D. (1980) Initial characterization of monoclonal antibodies against human monocytes. Proe. Natl. Acad. Sci. U.S.A. 77, 6764.