brain dissociation and seeding of cortical neurons in cell culture 

 Protocolo de cocultivo primario de microglía y neurona para estudios de interacción con el SNC

Microglia are tissue-resident macrophages that facilitate immunosurveillance and homeostasis in the central nervous system (CNS)1,2,3. They originate from yolk sac erythromyeloid progenitor cells that colonize the brain during embryonic development4,5,6&#160;and are maintained throughout the organism&#39;s life span through self-renewal, which involves proliferation and apoptosis7. At steady-state, resting microglia have ramified morphology and engage in tissue surveillance8,9,10.
Microglia express numerous cell-surface receptors, which enables them to rapidly respond to changes in the CNS11,12 and to promote inflammatory responses in the event of infections or tissue injury12,13,14, as well as during neurodegenerative diseases9,15, such as&#160;multiple sclerosis (MS)16,17. Microglia also express receptors to various neurotransmitters and neuropeptides18,19,20, which suggests they may also respond to and regulate neuronal activity21,22. Indeed, microglia and neurons interact in various forms of bidirectional communication8,23 such as direct interactions mediated by membrane proteins or indirect interactions through soluble factors or intermediate cells23,24.
For instance, various neurotransmitters secreted by neurons can modulate the neuroprotective or inflammatory activity of microglia25,26,27. Additionally, direct interactions between neurons and microglia help to maintain microglia in a homeostatic state28. Conversely, direct interactions of microglia with neurons can shape neuronal circuitry29 and influence neuronal signaling30,31,32. As disruptions of these interactions induce hyperexcitability of neurons30 and&#160;microglia reactivity33,34, dysregulated microglia-neuronal interactions&#160;are implicated&#160;as a contributing factor to&#160;neurological diseases33,35. Indeed, psychotic23,26 and neurodegenerative diseases have been described to exhibit dysfunctional microglia-neuronal interactions33. While these observations highlight the importance of microglia-neuronal communication in the CNS, specific mechanisms of how these interactions regulate microglial and neuronal functions in health and disease are relatively unknown.
Within a complex milieu such as the CNS, multiple environmental factors can influence microglia-neuronal interactions, which limits the ability to study transient cellular interactions in vivo. Here, we present an in vitro microglia-neuronal co-culture system that can be used to study direct cellular interactions between microglia and neurons. This protocol describes the generation of primary microglia and neurons from the cortices of neonatal mice between post-natal days 0-2 and embryonic mice days 16-18, respectively. Neurons and microglia are then co-cultured in 96-well plates for downstream high-throughput experiments. We previously used this approach to demonstrate that microglia phagocytosis protects neurons from oxidized phosphatidylcholine mediated cell death37, suggesting that this method can help to understand the roles of microglia in the context of neurodegeneration and MS. Similarly, microglia-neuronal co-cultures may also be useful for investigating the impact of microglia-neuronal crosstalk in other contexts such as viral infections38 or neuronal injury and repair39. Overall, in vitro microglia-neuronal co-culture systems&#160;enable researchers to study microglia-neuronal interactions in a manipulatable and controlled environment, which complements in vivo models.

Autor destacado: Modelo de cocultivo in vitro para el estudio de las interacciones microglía-neuronas en enfermedades

Generación y cocultivo de microglía primaria murina y neuronas corticales

Our research aims to investigate the interactions between neurons and microglia during disease conditions such as multiple sclerosis. We aim to recapitulate microglia neuronal interactions in vitro to better understand their functional importance in the CNS. We previously demonstrated that microglia phagocytosis protects neurons from oxidized phosphatidylcholine-mediated cell death with this co-culture.While other co-culture systems are also employed, this mixed culture system enables direct and close contact between microglia and neurons. In addition, it can remain healthy from 12 days to potentially three weeks, allowing for long-term studies of neuronal microglia interactions. Our protocol offers a simple and accessible method to co-culture microglia neurons that are easily manipulatable and can be further used in downstream assays to determine cellular functional changes.

Disociación cerebral y siembra de neuronas corticales en cultivo celular 

brain-dissociation-and-seeding-of-cortical-neurons-in-cell-culture

Research

JoVE Journal

Neuroscience

Brain Dissociation and Seeding of Cortical Neurons in Cell Culture 

43 Views. To begin, mince the isolated brains into pieces in the 50-milliliter conical tube with a sterile disposable scalpel blade. Add trypsin to the tube at a final concentration of 0.25%Immerse the tube in a 35 to 38 degrees Celsius hot water bath for 15 minutes. Gently pipette the tissue suspension up and down to dissociate cells.Then add 0.5 milliliters of fetal bovine serum to the homogenized suspension to neutralize trypsin activity. Filter the cell suspension through the 70-micrometer n...

Video: Brain Dissociation and Seeding of Cortical Neurons in Cell Culture 

roux-en-y gastric bypass operation in rats

Roux-en-Y Gastric Bypass Operation in Rats

Numerous studies using gastric bypass rat models have been recently conducted to uncover the underlying physiological mechanisms of Roux-en-Y gastric bypass operations. This article aims to demonstrate and discuss the technical and experimental details of our published gastric bypass rat model to understand advantages and limitations of this experimental...

robotic central pancreatectomy with roux-en-y pancreaticojejunostomy

Robotic Central Pancreatectomy with Roux-en-Y Pancreaticojejunostomy

Robotic central pancreatectomy may be used in selected patients in experienced centers. This protocol presents all steps and the feasibility of a robotic central pancreatectomy with Roux-en-Y pancreaticojejunostomy in a 16-year-old adolescent patient.

a protocol for roux-en-y gastric bypass in rats using linear staplers

A Protocol for Roux-en-Y Gastric Bypass in Rats using Linear Staplers

Roux-en-Y gastric bypass (RYGB) is performed to treat obesity and diabetes. However, the mechanisms underlying RYGB's efficacy are not fully understood, and studies are limited by technical difficulty leading to high mortality in animal models. This article provides instructions on how to perform RYGB in rats with high success...

techniques of sleeve gastrectomy and modified roux-en-y gastric bypass in mice

Techniques of Sleeve Gastrectomy and Modified Roux-en-Y Gastric Bypass in Mice

Bariatric surgery is the most efficient way to reduce body weight and the deadly metabolic complications (diabetes, obesity, and dyslipidemia) frequently associated with morbid obesity. Mouse models of bariatric surgery represent a unique asset for deciphering molecular mechanisms behind the beneficial effects of these surgeries on diabetes, hypertension, and...

roux-en-y gastric bypass procedure: a bariatric surgical procedure for gastric resectioning in murine model

Externalize the intestine and measure 8 centimeters from the pyloris. Using 5-0 non-absorbable sutures, place two ligatures on the intestine on either side of the 8-centimeter mark, and cut the tissue between the...

Roux-en-Y Gastric Bypass Procedure: A Bariatric Surgical Procedure for Gastric Resectioning in Murine Model

This video describes a bariatric surgery-Roux-en-Y procedure. This involves the cutting of the jejunum- the middle portion of the intestine- to generate two intestinal limbs. The biliary limb is attached to the existing intestinal loop and the alimentary limb is attached to the stomach, creating a gastric bypass. The gastric bypass created here helps to decrease the nutrient uptake and efficiently reduces body...

the subventricular zone en-face: wholemount staining and ependymal flow

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain.  Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis.  Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal...

en face preparation of mouse blood vessels

En Face Preparation of Mouse Blood Vessels

A procedure for making en face preparations of the mouse carotid artery and aorta is described. Such preparations, when immunofluorescently stained with specific antibodies, enable us to study localization of proteins and identification of cell types within the entire vascular wall by confocal...

En Face Preparation of Mouse Blood Vessels

using en face immunofluorescence staining to observe vascular endothelial cells directly

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

Here, we present a protocol for immunofluorescence staining to observe the endothelial cells of the mouse aorta directly. This technique is useful when studying the cellular and molecular phenotype of endothelial cells in different flow patterns and in the development of...

de novo generation of somatic stem cells by yap/taz

De Novo Generation of Somatic Stem Cells by YAP/TAZ

Availability of somatic SCs is crucial for regenerative medicine, disease modeling and to gain insight into SC properties. Here we present experimental strategies to reprogram, in vitro, differentiated adult cells into their corresponding expandable tissue-specific stem/progenitor cells by the transient expression of the single transcriptional co-activator...

De Novo Generation of Somatic Stem Cells by YAP/TAZ

assessment of de novo protein synthesis rates in caenorhabditis elegans

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Here, we introduce and describe a nonradioactive and noninvasive method to assess de novo protein synthesis in vivo, utilizing the nematode Caenorhabditis elegans and fluorescence recovery after photobleaching (FRAP). This method can be combined with genetic and/or pharmacological screens to identify novel modulators of protein...

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

sampling of n-methyl-n-nitrosourea-induced rat's retina damage and multi-index evaluation of pathological changes

This protocol describes a procedure for rat retinal sampling and utilizing hematoxylin-eosin staining, TUNEL assay, and Western blot to detect pathological changes and apoptosis in the retina after intraperitoneal injection of...

Sampling of N-methyl-N-nitrosourea-induced Rat's Retina Damage and Multi-index Evaluation of Pathological Changes

application of the en bloc concept combined with anatomic resection in laparoscopic hepatectomy

Application of the En Bloc Concept Combined with Anatomic Resection in Laparoscopic Hepatectomy

Numerous studies have demonstrated the advantages of anatomic resection. Nonetheless, whether anatomic resection can increase R0 resection rates remains controversial. Consequently, the present study describes an innovative procedure involving the en bloc concept combined with anatomic resection in laparoscopic hepatectomy, which can reduce postoperative recurrence and metastasis.

testing drosophila olfaction with a y-maze assay

Testing Drosophila Olfaction with a Y-maze Assay

We present a choice test to reveal the influence of odorants on Drosophila behavior using a Y-maze...

Testing Drosophila Olfaction with a Y-maze Assay

en face endocardial cushion preparation for planar morphogenesis analysis in mouse embryos

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

Classically, the endocardium of the mouse embryonic valve primordium has been analyzed using transversal, coronal, or sagittal sections. Our novel approach for en face, two-dimensional imaging of the endocardium at valvulogenic regions allows planar polarity and cell rearrangement analysis of the endocardium during valve...

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

isolation and cannulation of cerebral parenchymal arterioles

Isolation and Cannulation of Cerebral Parenchymal Arterioles

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular...

performing microscope-mounted y-shaped cutting tests

Performing Microscope-Mounted Y-Shaped Cutting Tests

Y-shaped cutting measures fracture-relevant length scales and energies in soft materials. Previous apparatuses were designed for benchtop measurements. This protocol describes the fabrication and use of an apparatus that orients the setup horizontally and provides the fine positioning capabilities necessary for in situ viewing, plus failure quantification, via an optical...

targeted microinjection and electroporation of primate cerebral organoids for genetic modification

Author Spotlight: Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification  

The electroporation of primate cerebral organoids provides a precise and efficient approach to introduce transient genetic modification(s) into different progenitor types and neurons in a model system close to primate (patho)physiological neocortex development. This allows the study of neurodevelopmental and evolutionary processes and can also be applied for disease...

Genetic Modification of Primate Cerebral Organoids

live imaging of primary cerebral cortex cells using a 2d culture system

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and...

preparation of n-(2-alkoxyvinyl)sulfonamides from n-tosyl-1,2,3-triazoles and subsequent conversion to substituted phthalans and phenethylamines

Preparation of N-2-alkoxyvinylsulfonamides from N-tosyl-1,2,3-triazoles and Subsequent Conversion to Substituted Phthalans and Phenethylamines

Representative experimental procedures for the synthesis of N-(2-alkoxyvinyl)sulfonamides and subsequent conversion to phthalan and phenethylamine derivatives are presented in...

Preparation of N-(2-alkoxyvinyl)sulfonamides from N-tosyl-1,2,3-triazoles and Subsequent Conversion to Substituted Phthalans and Phenethylamines

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS ...

Investigación

Neurociencias

Brain Isolation from Neonatal Mice for Primary Microglia Culture

To begin, add 5 milliliters of 10 micrograms per milliliter Poly-L-ornithine hydrobromide to each T75 flask. Incubate at 37 degrees Celsius in a 5%carbon dioxide incubator for at least one hour. After decapitating the mouse, gently hold the head and create a midline incision in the cranium, starting from the neck to the nose.Peel the skull back with tissue forceps to reveal the brain. To scoop the brain out, insert the tissue forceps under the brain, and lift the brain out from the head. After placing the brain into a Petri dish containing 20 milliliters of Leibovitz's L-15 media under a dissection microscope, using a pair of micro-forceps, carefully remove the brainstem and the meninges.Collect the brains in a 50-milliliter conical tube containing 5 milliliters of Leibovitz's L15 media.

Aislamiento cerebral de ratones neonatos para cultivo primario de microglía

Preparing and Maintaining Mixed Glia Culture Derived from Neonatal Mice Brains

To begin, mince the isolated brains into approximately one-square-millimeter pieces with a sterile disposable scalpel blade. Carefully transfer the minced brains into a new sterile 50 milliliter tube. Add a final concentration of 0.25%trypsin to the minced brains.Incubate the tissue in a 37-degrees-Celsius water bath for 20 minutes. Add five milliliters of complete glia culture media to neutralize the trypsin and gently mix the tissue suspension using a 10 milliliter pipette. Carefully decant the cell suspension through the cell strainer.Then remove the plunger from a sterile one-milliliter syringe and grind the tissue into the mesh using the plunger. Add equal volumes of cell suspension containing one to two brains with glia culture media to each flask resulting in a final volume of 15 milliliters. Sparse cells and excessive cellular debris were observed on day one.By day four, adherent astrocytes were generated. Confluent cells with typical astrocyte morphology were observed by day eight of culture.

Preparación y mantenimiento de un cultivo mixto de glía derivado de cerebros de ratones neonatos

Isolating Brains from E16-E18 Mouse Embryos for Primary Neuron Culture

Begin by coating T25 flasks with five milliliters of 10 micrograms per milliliter poly-L-ornithine, and incubate them. To isolate the brain from mouse embryos, lift the skin of the euthanized pregnant mouse's lower abdomen with tissue forceps. Using dissection scissors, perform a V-shaped incision from this point to the lower rib cage.Grab the uterine horns containing the embryos with tissue forceps, and gently remove the embryos from the abdominal cavity. Using spring scissors, create a midline incision at the top of the skull, starting from the back of the neck to the nose. Lift the skull away from the incision site to reveal the brain.Gently scoop out the brain with tissue forceps and transfer the brain to a 10 centimeter Petri dish containing 15 milliliters of Hanks'balanced salt solution. Under a dissection microscope, carefully remove the brainstem and the meninges on both sides of the brain cortices with a pair of micro forceps.

Aislamiento de cerebros de embriones de ratón E16-E18 para el cultivo de neuronas primarias

To begin, mince the isolated brains into pieces in the 50-milliliter conical tube with a sterile disposable scalpel blade. Add trypsin to the tube at a final concentration of 0.25%Immerse the tube in a 35 to 38 degrees Celsius hot water bath for 15 minutes. Gently pipette the tissue suspension up and down to dissociate cells.Then add 0.5 milliliters of fetal bovine serum to the homogenized suspension to neutralize trypsin activity. Filter the cell suspension through the 70-micrometer nylon mesh cell strainer. Using a one-milliliter syringe plunger, gently grind the tissue into the cell strainer.Then centrifuge the conical tube at 300 g for five minutes at four degrees Celsius. Discard the Poly-L-Ornithine in the T25 flasks and rinse the flasks three times with five milliliters of PBS. Carefully decant the tube to remove the supernatant and resuspend the cell pellet by gently pipetting with two to three milliliters of complete neurobasal media.Plate the cells in the Poly-L-Ornithine-coated T25 flasks with approximately 20 million cells per flask in five milliliters of complete neurobasal media. Incubate the cell culture at 37 degrees Celsius in a 5%carbon dioxide incubator. Neurons in T25 flasks and 96-well plates adhered and formed neurite processes by day three.Suboptimal neuron cultures contained cell clumps and debris.

Co-Culture of Primary Neurons and Microglia for Studying Microglia-Neuron Interactions

To begin, take neurons grown in T25 flasks for co-culture. Replace the media with five milliliters of versene solution containing trypsin and Dnase I.Add 0.5 milliliters of fetal bovine serum to neutralize the trypsin and Dnase I.Transfer cells to a 15 milliliter conical tube. Wash the flask with five milliliters of complete neurobasal media once to maximize the collected neurons.Then, centrifuge the cell suspension at 300 G for five minutes at four degrees celsius. After discarding the supernatant, gently resuspend the cell pellet in two to three milliliters of complete neurobasal media. Seed 100 microliters of the cell suspension to each well to obtain 75, 000 neurons per well, between eight to 10 days of mixed glia cultivation.Using a 10 milliliter pipette, gently wash the T75 flasks with glia culture media to collect microglia and transfer the cells and media into a sterile 50 milliliter conical tube. Centrifuge the cell collection at 300 G for five minutes at four degrees celsius. Once the supernatant is removed, gently resuspend the pellet in two milliliters of complete neurobasal media.From the 96-well neuronal culture plate, remove 100 microliters of neurobasal media and add 100 microliters of 250, 000 cells per milliliter cell suspension. In co-culture round and large microglia were observed on top or between neurons and neurite processes.