Isolation and Culture of Mouse Cortical Astrocytes

Schildge, Sebastian; Bohrer, Christian; Beck, Kristina Barbara; Schachtrup, Christian

doi:10.3791/50079

Cited by 341 publications

(299 citation statements)

References 36 publications

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“…Especially the immature astrocyte expression rate of GFAP does not exceed 40% under physiological conditions 28 . Depending on the age-and type-specific use of astrocytes, other markers of choice can be considered such as GLAST, ALDH1L1, BLBP, Aquaporin-4 and S100B 29 .…”

Section: Discussionmentioning

confidence: 99%

Sex Differences in Mouse Hippocampal Astrocytes after <em>In-Vitro</em> Ischemia

Chanana

Tümtürk

Kintner

et al. 2016

JoVE

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Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 99%

Sex Differences in Mouse Hippocampal Astrocytes after <em>In-Vitro</em> Ischemia

Chanana

Tümtürk

Kintner

et al. 2016

JoVE

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“…The protocol presented here is based on the work of McCarthy and de Vellis 19 , and a very similar detailed protocol for mouse astrocytes is available 20 . To generate primary cultures of cortical astrocytes from embryonic (E18) rat brains, a pregnant rat needs to be sacrificed, the embryos need to be harvested from the uterus, and the brains need to be isolated from the embryos.…”

Section: Notementioning

confidence: 99%

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Frega

Gestel

Linda

et al. 2017

JoVE

119

189

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Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-2 12. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (>95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of celltype specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

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“…Cortical astrocytes from 2-to 4-day-old WT and CerS2 null mice (PewznerJung et al, 2010b) were isolated as described (Schildge et al, 2013). Astrocytes were grown to 95% confluence for 7-10 days, other cells were removed from the dishes by vigorous shaking (2 h, 200 rpm on an orbital shaker), and astrocytes were replated and grown to 95% confluence for up to 23 days.…”

Section: Astrocyte Culturesmentioning

confidence: 99%

Oxidative stress elicited by modifying the ceramide acyl chain length reduces the rate of clathrin-mediated endocytosis

Volpert

Ben‐Dor

Tarcic

et al. 2017

Journal of Cell Science

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Sphingolipids modulate clathrin-mediated endocytosis (CME) by altering the biophysical properties of membranes. We now examine CME in astrocytes cultured from ceramide synthase 2 (CerS2) null mice, which have an altered sphingolipid acyl chain composition. The rate of endocytosis of low-density lipoprotein and transferrin, which are internalized via CME, was reduced in CerS2 null astrocytes, although the rate of caveolin-mediated endocytosis was unaltered. Levels of clathrin heavy chain were increased, which was due to decreased levels of Hsc70 (also known as HSPA8), a protein involved in clathrin uncoating. Hsc70 levels were decreased because of lower levels of binding of Sp1 to position −68 in the Hsc70 promoter. Levels of Sp1 were downregulated due to oxidative stress, which was elevated fourfold in CerS2 null astrocytes. Furthermore, induction of oxidative stress in wild-type astrocytes decreased the rate of CME, whereas amelioration of oxidative stress in CerS2 null astrocytes reversed the decrease. Our data are consistent with the notion that sphingolipids not only change membrane biophysical properties but also that changes in their composition can result in downstream effects that indirectly impinge upon a number of cellular pathways, such as CME.

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Isolation and Culture of Mouse Cortical Astrocytes

Cited by 341 publications

References 36 publications

Sex Differences in Mouse Hippocampal Astrocytes after <em>In-Vitro</em> Ischemia

Sex Differences in Mouse Hippocampal Astrocytes after <em>In-Vitro</em> Ischemia

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Oxidative stress elicited by modifying the ceramide acyl chain length reduces the rate of clathrin-mediated endocytosis

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