Characterization of an improved procedure for the removal of microglia from confluent monolayers of primary astrocytes

Hamby, Mary E.; Uliasz, Tracy F.; Hewett, Sandra J.; Hewett, James A.

doi:10.1016/j.jneumeth.2005.06.016

Cited by 98 publications

(124 citation statements)

References 43 publications

(51 reference statements)

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“…Microglia also can be removed by adding reagents such as L-leucine methyl ester, a lysosomotrophic agent destroying phagocytic cells (Esen et al, 2004;Thiele et al, 1983). This is sometimes combined with antimitotic drugs such as cytosine arabinoside (Ara-C), preventing further proliferation of microglia (Hamby et al, 2006). Others have specifically depleted microglia using antibody-conjugated saporin toxin (Montero et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

Microglia are required for astroglial toll‐like receptor 4 response and for optimal TLR2 and TLR3 response

Holm

Dræby

Owens

2012

Glia

111

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Within the central nervous system, astrocytes and microglia are the primary responders to endogenous ligands released upon injury and stress, as well as to infectious pathogens. Toll-like receptors (TLRs) are implicated in recognition of both types of stimulus. Whether astrocytes respond as strongly as microglia to TLR agonists remains contentious. In this study, we have rigorously purified astrocytes to determine their capacity for autonomous TLR response, in absence of microglia. We used flow cytometry and differential adhesion as well as a myeloid lineage-specific suicide gene to purify astrocytes from mixed glial cultures and measured their response to TLR agonists. Our results show that the response of astrocytes to TLR2 and TLR3 agonists is greatly enhanced by, and response to TLR4 agonists is completely dependent on, the presence of functional microglia. In the case of the TLR4 response to lipopolysaccharide, microglia exert their effect on astrocytes at least partially through release of soluble mediators that directly activate or facilitate astrocyte responses. Our findings underline the contribution of glial crosstalk in CNS responses to injury or inflammation. V

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Section: Discussionmentioning

confidence: 99%

Microglia are required for astroglial toll‐like receptor 4 response and for optimal TLR2 and TLR3 response

Holm

Dræby

Owens

2012

Glia

111

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“…Cells were then placed in maintenance media (see above), which was exchanged once per week until experimentation. In most cases, cultures highly enriched for astrocytes were generated by removing residual microglia by treatment with 50 mM L-leucine methyl ester for 30 min, 1 day prior to experimentation (45). Experiments were performed on cultures 21-35 days in vitro.…”

Section: Methodsmentioning

confidence: 99%

Interleukin 1β Regulation of the System xc− Substrate-specific Subunit, xCT, in Primary Mouse Astrocytes Involves the RNA-binding Protein HuR

Shi

Hewett

et al. 2016

Journal of Biological Chemistry

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“…Primary murine cortical astrocyte cultures were prepared as described previously (33). In brief, cortices were harvested from 1-day-old mice under deep isofluorane anesthesia.…”

Section: Isolation and Preparation Of Murine Primary Cellsmentioning

confidence: 99%

Neuroprotective Immunity: T Cell-Derived Glutamate Endows Astrocytes with a Neuroprotective Phenotype

Garg

Banerjee

Kipnis

2008

The Journal of Immunology

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A well-controlled T cell response to CNS injury may result in increased neuronal survival. However, the precise mechanism of T cell-induced neuroprotection is unknown. In this study, we report the unexpected finding that during culture of T cells, high levels of glutamate accumulate, which are efficiently cleared if T cells are cocultured with astrocytes. The T cell-derived glutamate elicits in turn, the release of neuroprotective thiols (cysteine, glutathione, and cysteinyl-glycine) and lactate from astrocytes. Media obtained from astrocytes conditioned in the presence of T cells reduce neuronal apoptosis induced by oxidative stress in primary neuronal cultures from 48 ± 14 to 9 ± 4% (p < 0.001). Inhibition of glutamate-dependent signaling during astrocyte-T cell cocultivation by a glutamate uptake inhibitor, l-aspartic acid β-hydroxamate, abolishes this neuroprotective effect. The ability of astrocytes to clear extracellular glutamate is impaired under conditions of oxidative stress. We demonstrate that T cells, via secreted cytokines, restore glutamate clearance capacity of astrocytes under oxidative conditions. Furthermore, under normoxic conditions, glutamate-buffering capacity of astrocytes is increased upon cocultivation with T cells. It is known that, following CNS injury, astrocytes can respond with beneficial or destructive effects on neurons. However, the context and signaling mechanisms for this dual astrocytic response are unknown. Our results implicate T cells as potential determinants of the context that elicits a protective role for astrocytes in the damaged CNS.

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Characterization of an improved procedure for the removal of microglia from confluent monolayers of primary astrocytes

Cited by 98 publications

References 43 publications

Microglia are required for astroglial toll‐like receptor 4 response and for optimal TLR2 and TLR3 response

Microglia are required for astroglial toll‐like receptor 4 response and for optimal TLR2 and TLR3 response

Interleukin 1β Regulation of the System xc− Substrate-specific Subunit, xCT, in Primary Mouse Astrocytes Involves the RNA-binding Protein HuR

Neuroprotective Immunity: T Cell-Derived Glutamate Endows Astrocytes with a Neuroprotective Phenotype

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