eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Primary cell culture preparation
<ol>
	<li>Brain tissue isolation 
	NOTE: Isolation should be performed on ice, using ice cold solutions.
	<ol>
		<li>Use newborn pups 1-3 days old for primary neonatal cell culture. 
		&#8203;NOTE: For the study presented here, Sprague Dawley hSOD1G93A and non-transgeni...

generating a primary culture of astrocytes from a rat pup brain

Source: Mili&#263;evi&#263;, K., et al., <a href="https://app.jove.com/v/63483">Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis.</a> J. Vis. Exp. (2022)
The video demonstrates a technique for culturing primary astrocytes from the rat cortex. The cortex is mechanically dissociated to obtain a homogeneous glial cell suspension, which is then plated on polypeptide-coated plates to promote selective proliferation of astrocytes. Subsequently, the astrocytes are enzymatically dissociated from the plates and transferred to fresh media, allowing the culture of pure astrocytes.

Generating a Primary Culture of Astrocytes from a Rat Pup Brain

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Primary cell culture preparation

...

Add a cortex from a rat pup brain to a tube with a buffer.
With a pipette, gently move the cortex up and down to break it into small tissue pieces.
Centrifuge and discard the supernatant.
Resuspend the pieces in a culture medium.
Pass the pieces through needles of decreasing diameters to obtain a single-cell suspension.
Transfer the cells to a polypeptide-coated plate containing the culture medium and incubate.
Astrocytes strongly adhere to polypeptide-coated surfaces, facilitating their growth over other cells.
Remove the medium and wash with a buffer to remove any loosely attached cells.
Treat with a digestive enzyme to detach the astrocytes from the plate.
Add the medium to stop the enzymatic activity.
Collect the supernatant in a tube, and centrifuge.
Discard the supernatant and then resuspend the cells in a fresh culture medium.
Place the cells into a plate and incubate to facilitate the multiplication of astrocytes.

generating-a-primary-culture-of-astrocytes-from-a-rat-pup-brain

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

JoVE Journal

Live-imaging of Mitochondrial System in Cultured Astrocytes

While much attention has been given to mitochondrial alterations at the neuronal level, recent evidence demonstrates that mitochondrial dynamics and function in astrocytes are implicated in cognition. This article describes the method for time-lapse imaging of astrocyte cultures equipped with a mitochondrial biosensor: MitoTimer. MitoTimer is a powerful and unique tool to assess mitochondrial dynamics, mobility, morphology, biogenesis, and redox state. Here, the different procedures for culture, image acquisitions, and subsequent mitochondrial analysis are presented.

This article describes the method for mitochondrial time-lapse imaging of astrocyte cultures equipped with MitoTimer biosensor and the resulting multiparametric analysis of mitochondrial dynamics, mobility, morphology, biogenesis, redox state, and turnover.

While much attention has been given to mitochondrial alterations at the neuronal level, recent evidence demonstrates that mitochondrial dynamics and ...

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro&#160;experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...

Generating a Primary Culture of Astrocytes from a Rat Pup Brain

Transcript