Abstract
Primary rodent astroglial-enriched cultures are the most popular model to study astroglial biology in vitro. From the original methods described in the 1970's a great number of minor modifications have been incorporated into these protocols by different laboratories. These protocols result in cultures in which the astrocyte is the predominant cell type, but astrocytes are never 100% of cells in these preparations. The aim of this review is to bring attention to the presence of microglia in astroglial cultures because, in my opinion, the proportion of and the role that microglial cells play in astroglial cultures are often underestimated. The main problem with ignoring microglia in these cultures is that relatively minor amounts of microglia can be responsible for effects observed on cultures in which the astrocyte is the most abundant cell type. If the relative contributions of astrocytes and microglia are not properly assessed an observed effect can be erroneously attributed to the astrocytes. In order to illustrate this point the case of NO production in activated astroglial-enriched cultures is examined. Lipopolysaccharide (LPS) induces nitric oxide (NO) production in astroglial-enriched cultures and this effect is very often attributed to astrocytes. However, a careful review of the published data suggests that LPS-induced NO production in rodent astroglial-enriched cultures is likely to be mainly microglial in origin. This review considers cell culture protocol factors that can affect the proportion of microglial cells in astroglial cultures, strategies to minimize the proportion of microglia in these cultures, and specific markers that allow the determination of such microglial proportions.
Highlights
Our knowledge of the functional potentialities of astrocytes has not ceased to grow in the last decades
Several authors have noted previously that caution must be used when making a claim of pure astroglial cultures [22,26,52] and that attention must be paid to the presence of microglia in these cultures [66,67]
In my opinion it is necessary that authors, referees and editors become aware of this question in order to reduce the number of publications in which the presence of microglia in astroglial-enriched cultures is ignored and especially those that attribute to astrocytes roles that are played by contaminating cells, microglia
Summary
Several authors have noted previously that caution must be used when making a claim of pure astroglial cultures [22,26,52] and that attention must be paid to the presence of microglia in these cultures [66,67]. Those interested in working with highly-enriched astroglial cultures should use an adequate protocol to reduce the proportion of microglia, e.g. laminin coating, shaking, subculture at low density, L-leucine methyl ester, frequent medium changes. High resolution techniques (e.g. immunocytochemistry, in situ hybridisation) should be used to clearly attribute an observed effect to a given cell type in astroglial cultures The use of both highly-enriched astroglial and microglial cultures can help in this respect. If these approaches are not possible the question should be left open and say, for example, "LPS induced an increase of X in astroglial-enriched cultures" which is a true observation instead of "LPS induced an increase of X in astrocytes" which could be a false deduction/misinterpretation. Ara-C, Cytosine arabinoside DiI-Ac-LDL, DiI labelled acetylated-Low Density Lipoprotein GFAP, glial fibrillary acidic protein GSA, Griffonia simplicifolia agglutinin Iba, Ionized calcium binding adaptor molecule 1 IFNγ, interferon-γ LPS, Lipopolysaccharide NOS2, NO synthase-2 RCA, Ricinus Communis Agglutinin TGFβ1, transforming growth factor β1
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