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Journal of Neuroscience, Vol 9, 183-194, Copyright © 1989 by Society for Neuroscience
Lipopolysaccharide-free conditions in primary astrocyte cultures allow growth and isolation of microglial cells
PJ Gebicke-Haerter, J Bauer, A Schobert and H Northoff
Pharmacological Institute, University of Freiburg, Medical School, Federal Republic of Germany.
Primary rat astrocyte cultures were used to isolate a macrophage population
that does not adhere to the confluent glial cells. The cells multiplied
vigorously in coculture with astrocytes during the 14 d culture period,
provided that functionally active lipopolysaccharide (LPS) was either
absent or present in very low concentrations. Based on morphological,
immunocytochemical, and pharmacological data, it was concluded that the
isolated cells were microglia, the resident macrophages of the brain. The
findings characterized them as a distinct cell population that shares
features both of peritoneal macrophages and of astroglial cells. Like
peritoneal macrophages, the isolated cells were able to phagocytize as
shown by their ingestion of latex beads and uptake of L-leucyl methylester.
Furthermore, they were immunocytochemically stainable by a specific
monoclonal antibody (ED 1) against a macrophage-specific antigen (Dijkstra
et al., 1985). They also synthesized prostaglandin E2 (PGE2) and secreted
interleukin 1 (IL- 1) upon stimulation with LPS. Upon stimulation with the
ionophore A23187, PGD2, the predominant prostaglandin of the brain, was the
major PG metabolite released by these cells. In contrast to peritoneal
macrophages, microglial cells were able to multiply. Proliferation of
microglial cells in coculture with astrocytes was suppressed when 2 ng
LPS/ml or higher concentrations were added to astroglial culture media.
These astrocyte cultures, which contained approximately 1% microglia, were
used to investigate the influence of LPS on prostaglandin and IL-1
secretion in order to compare astroglial and microglial features.
Increasing LPS concentrations induced increased PGE2 secretion, whereas
PGD2 secretion was essentially unaffected by LPS. The critical influence of
LPS contaminations in most of the commercially available animal sera used
for astrocyte cultures on cellular composition in general and on metabolism
of hormones and growth factors in particular is discussed.
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