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Lipopolysaccharide (LPS)-free conditions allow growth and purification of postnatal brain macrophages (microglia)
Journal of Immunological Methods, 116 (1989) 147 Elsevier
147
JIM 05071
Letter to the editors
Lipopolysaccharide (LPS)-free conditions allow growth and purification of postnatal brain macrophages (microglia) H. N o r t h o f f a, j. Bauer 2, A. Schobert 3, W.A. Flegel 1 a n d P.J. G e b i c k e - H a e r t e r 3 I D RK-Blutspendezentrale Ulm, Oberer Eselsberg 10, D-7900 Ulm, F.R.G., 2 Department of Internal Medicine II, University Freibur~ Hugstetter Strasse 55, D-7800 Freibur~ F.R.G., and 3 Pharmacological Institute, University Freiburg, Herman-Herderstrasse 5, D-7800 Freiburg, F.R. G. (Received 3 October 1988, accepted 10 October 1988)
Dear Editors, Recently we pointed out that sera used to supplement cell cultures should be tested for functionally active endotoxin (lipopolysaccharide, LPS), e.g., by an assay system involving interleukin-1 (IL-1) induction and assay (IPAS) (Northoff et al., 1986). Applying this strategy to neural cell cultures, we found some unexpected results that may be of interest to workers outside the field of neuroscience research. Astrocyte cultures have been described in the literature as being normally contaminated with 1% of microglia, the resident macrophage of the brain. Attempts to obtain enriched microglial cell cultures have generally proved to be unrewarding. Two methods published recently (Ling et al., 1983; Giulian and Baker, 1986) based on differential adhesion, are time-consuming, and result in no more than 95% purity at best. Moreover, the yield of enriched cells is low since they do not proliferate under the 'standard' conditions used for astrocyte cultures. We have found recently, however, that microglia did multiply in rat astrocyte cultures provided the supplementary sera were IPAS negative and that LPS contaminations of culture media from other sources (water, glassware, etc.) were rigorously excluded. The addition of 2 ng L P S / m l or more reversed this effect, whereas astrocyte growth appeared to be unabated. Interestingly, microglia proliferating under LPS-free conditions detached Correspondence to: H. Northoff, DRK-Blutspendezentrale Ulm, Oberer Eselsberg 10, D-7900 UIm, F.R.G.
from the astroglial cell layer and began to float. They could simply be 'scooped off' to yield a 100% pure cell population that attached within minutes to tissue culture plastic. When LPS was added to isolated microglia, they stopped replication immediately and responded with the production of IL-1 and prostaglandin E 2. LPS also increased the synthesis of apolipoprotein E and induced transcription of m R N A for nerve growth factor (NGF) in these cells. Further biochemical and morphological features of isolated microglia will appear in a specialized journal (GebickeHaerter et al., 1988). It appears that LPS-free culture conditions represent a powerful and simple strategy for obtaining a pure microglial cell population readily accessible to functional analysis. Moreover, the fact that LPS-free conditions allowed spontaneous growth of microglia may have its own significance. LPS not only induces mediator production but has the potential also to regulate growth in these ceils. Comparable cell culture systems used in unrelated areas of immunological or biological research might also prove to be sensitive to the effects of LPS.
References Gebicke-Haerter, P.J., Bauer, J., Schobert, A. and Northoff, H. (1988) J. Neurosci., in press. Giulian, D. and Baker, T.J. (1986) J. Neurosci. 6, 2163. Ling, E.A., Tseng, C.Y., Yoon, F.C.T. and Wong, W.C. (1983) J. Anat. 137, 223. Northoff, H., Kabelitz, D. and Galanos, C. (1986) Immunol. Today 7, 126.
0022-1759/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
147
JIM 05071
Letter to the editors
Lipopolysaccharide (LPS)-free conditions allow growth and purification of postnatal brain macrophages (microglia) H. N o r t h o f f a, j. Bauer 2, A. Schobert 3, W.A. Flegel 1 a n d P.J. G e b i c k e - H a e r t e r 3 I D RK-Blutspendezentrale Ulm, Oberer Eselsberg 10, D-7900 Ulm, F.R.G., 2 Department of Internal Medicine II, University Freibur~ Hugstetter Strasse 55, D-7800 Freibur~ F.R.G., and 3 Pharmacological Institute, University Freiburg, Herman-Herderstrasse 5, D-7800 Freiburg, F.R. G. (Received 3 October 1988, accepted 10 October 1988)
Dear Editors, Recently we pointed out that sera used to supplement cell cultures should be tested for functionally active endotoxin (lipopolysaccharide, LPS), e.g., by an assay system involving interleukin-1 (IL-1) induction and assay (IPAS) (Northoff et al., 1986). Applying this strategy to neural cell cultures, we found some unexpected results that may be of interest to workers outside the field of neuroscience research. Astrocyte cultures have been described in the literature as being normally contaminated with 1% of microglia, the resident macrophage of the brain. Attempts to obtain enriched microglial cell cultures have generally proved to be unrewarding. Two methods published recently (Ling et al., 1983; Giulian and Baker, 1986) based on differential adhesion, are time-consuming, and result in no more than 95% purity at best. Moreover, the yield of enriched cells is low since they do not proliferate under the 'standard' conditions used for astrocyte cultures. We have found recently, however, that microglia did multiply in rat astrocyte cultures provided the supplementary sera were IPAS negative and that LPS contaminations of culture media from other sources (water, glassware, etc.) were rigorously excluded. The addition of 2 ng L P S / m l or more reversed this effect, whereas astrocyte growth appeared to be unabated. Interestingly, microglia proliferating under LPS-free conditions detached Correspondence to: H. Northoff, DRK-Blutspendezentrale Ulm, Oberer Eselsberg 10, D-7900 UIm, F.R.G.
from the astroglial cell layer and began to float. They could simply be 'scooped off' to yield a 100% pure cell population that attached within minutes to tissue culture plastic. When LPS was added to isolated microglia, they stopped replication immediately and responded with the production of IL-1 and prostaglandin E 2. LPS also increased the synthesis of apolipoprotein E and induced transcription of m R N A for nerve growth factor (NGF) in these cells. Further biochemical and morphological features of isolated microglia will appear in a specialized journal (GebickeHaerter et al., 1988). It appears that LPS-free culture conditions represent a powerful and simple strategy for obtaining a pure microglial cell population readily accessible to functional analysis. Moreover, the fact that LPS-free conditions allowed spontaneous growth of microglia may have its own significance. LPS not only induces mediator production but has the potential also to regulate growth in these ceils. Comparable cell culture systems used in unrelated areas of immunological or biological research might also prove to be sensitive to the effects of LPS.
References Gebicke-Haerter, P.J., Bauer, J., Schobert, A. and Northoff, H. (1988) J. Neurosci., in press. Giulian, D. and Baker, T.J. (1986) J. Neurosci. 6, 2163. Ling, E.A., Tseng, C.Y., Yoon, F.C.T. and Wong, W.C. (1983) J. Anat. 137, 223. Northoff, H., Kabelitz, D. and Galanos, C. (1986) Immunol. Today 7, 126.
0022-1759/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)