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Disclaimer: &#160;All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical homogenization of the brain and spinal cord
NOTE: Volumes described in this section are enough for the homogenization of one-half brain or spinal cord.
<ol>
	<li>Pre-chill the glass mortar of the Dounce tissue grinder (Table of Materials) set on ice.</li>
	<li>Add 3 mL of pre-chilled 1x Hank&#39;s balanced salt solution (HBSS) to the mortar.</li>
	<li>Transfer the tissue (brain or spinal cord) from the well of the 6-well plate into the glass mortar, making sure it is dipped in 1x HBSS and sits at the bottom of the mortar.</li>
	<li>Gently smash the tissue with 10 strokes of pestle A followed by 10 strokes of pestle B. Transfer the homogenized mix into a new 15 mL conical tube.</li>
	<li>Fill the tube to a final volume of 10 mL by using pre-chilled 1x HBSS and centrifuge for 10 min at 320 x g at 4 &#176;C.</li>
	<li>Aspirate the supernatant and add ice-cold 1x HBSS to each tube up to a final volume of 7 mL and gently resuspend the pellet by vortexing.</li>
</ol>
2. Debris removal 
NOTE: Removal of debris, composed mainly of undigested tissue and myelin sheaths, is a critical step to allow efficient staining of the tissue homogenate for subsequent flow cytometric analyses.
<ol>
	<li>Filter each sample through a 70 &#181;m cell strainer to remove any undigested tissue chunks. This step is particularly important when working with spinal cord tissues, as these samples are more likely to contain undigested nerve fragments or meninges that could affect the subsequent steps.</li>
	<li>Make sure that the final volume is 7 mL in each sample tube. If not, fill with ice-cold 1x HBSS up to 7 mL.</li>
	<li>Add 3 mL of pre-chilled isotonic Percoll solution (IPS) to each sample to make a final volume of 10 mL of a solution containing density gradient medium at a 30% final concentration. Gently vortex the samples to ensure they are homogeneously mixed.</li>
	<li>Centrifuge samples for 15 min at 700 x g at 18 &#176;C, making sure to set the acceleration of the centrifuge to 7 and the brake to 0. 
	NOTE: Centrifugation should take approximately 30 min.</li>
	<li>Delicately remove the samples from the centrifuge. 
	NOTE: A whitish disk composed of debris and myelin should be visible floating on the surface of the solution. A pellet (containing the cells of interest) should be visible at the bottom of the tube.</li>
	<li>Carefully aspirate all the whitish disk of debris and then the rest of the supernatant, making sure not to dislodge the pellet. Leave about 100 &#181;L of solution on top of the cell pellet to avoid the risk of inadvertently dislodging it.</li>
	<li>Add 1 mL of flow cytometry (FACS) blocking (BL) solution, resuspend the pellet by pipetting up and down with a 1000 &#181;L pipette tip, and transfer samples to 1.5 mL tubes.</li>
	<li>Centrifuge for 5 min at 450 x g at room temperature (RT).</li>
	<li>Gently aspirate the supernatant and resuspend the pellet in the appropriate buffer compatible with downstream analyses</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10X HBSS (Calcium, Magnesium chloride, and Magnesium Sulfate-free)</td>
			<td>Gibco</td>
			<td>14185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>70 mm Cell Strainer</td>
			<td>Corning</td>
			<td>431751</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Tubes (15 mL)</td>
			<td>CellTreat</td>
			<td>229411</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Tubes (50 mL)</td>
			<td>CellTreat</td>
			<td>229422</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce Tissue Grinder set (Includes Mortar as well as Pestles A and B)</td>
			<td>Sigma-Aldrich</td>
			<td>D9063-1SET</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare</td>
			<td>10266569</td>
			<td>sold as not sterile reagent</td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>Sigma</td>
			<td>65455529</td>
			<td>sterile reagent (to be used for applications requiring sterility)</td>
		</tr>
		<tr>
			<td>Percoll PLUS</td>
			<td>Sigma</td>
			<td>GE17-5445-01</td>
			<td>reagent containing very low traces of endotoxin</td>
		</tr>
	</tbody>
</table>

isolation of multiple cell types from a mouse brain by mechanical homogenization

Source: Molina Estevez, F. J., et al. <a href="https://app.jove.com/v/60335">Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods.</a> J. Vis. Exp. (2019).
This video demonstrates the isolation of multiple types of cells from mouse brain tissue. Tissue is first mechanically sheared using a tissue grinder in a salt solution to maintain an osmotic balance. The tissue homogenate is then subjected to density gradient centrifugation to remove cellular debris. Cells are then resuspended in a solution for further analysis.

Isolation of Multiple Cell Types from a Mouse Brain by Mechanical Homogenization

Disclaimer: &#160;All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical ...

Begin by placing the tissue grinder components on ice. This device includes a mortar and two pestles of different diameters.
Pour a chilled salt buffer into the mortar and then add mouse brain tissue.
The buffer maintains osmotic balance, preserving cell structure and function.
Grind the tissue with multiple gentle strokes using the smaller diameter pestle, which mechanically shears the tissue into smaller pieces.
Next, stroke with the larger diameter pestle, facilitating further breakdown of tissue into its component cells.
Transfer the mixture to a tube and centrifuge.
Remove the supernatant.
Add chilled salt buffer, and vortex to break the cell clumps and the myelin.
Next, add a density gradient medium and centrifuge.
Due to differences in shape and mass, cells will settle in the lower layer, while debris and myelin accumulate in the top layer.
Discard the supernatant.
Add a suitable solution to obtain a multiple-cell suspension for further use.

isolation-multiple-cell-types-from-mouse-brain-mechanical

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

93 vistas. Fuente: Molina Estevez, F. J., et al. Caracterización Citométrica de Flujo Simultáneo de Múltiples Tipos de Células Recuperadas del Cerebro/Médula Espinal de Ratón a través de Diferentes Métodos de Homogeneización. J. Vis. Exp. (2019). Este video muestra el aislamiento de múltiples tipos de células a partir de tejido cerebral de ratón. El tejido se corta primero mecánicamente con un molinillo de papel en una solución salina para mantener un equilibrio osmótico. A continuación, ...

Video: Aislamiento de múltiples tipos de células del cerebro de un ratón mediante homogeneización mecánica - Concepto

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

Aislamiento de microglia específica de la región del hemisferio cerebral de un ratón adulto para la secuenciación profunda del ARN de una sola célula

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Investigación

JoVE Journal

Neurociencias

Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). Microglia can be divided into resting state and activated state and can rapidly change state in response to the microenvironment of the brain. Microglia will be activated under different pathological conditions and exhibit different phenotypes. In addition, there are many different subgroups of activated microglia and great heterogeneity between different subgroups. The heterogeneity mainly depends on the molecular specificity of microglia. Studies have revealed that microglia will be activated and play an important role in the pathological process of inflammatory demyelination. To better understand the characteristics of microglia in inflammatory demyelinating diseases such as multiple sclerosis and neuromyelitis optica spectrum disorder, we propose a perilesional primary microglial sorting protocol. This protocol utilizes columnar magnetic-activated cell sorting (MACS) to obtain highly purified primary microglia and preserve the molecular characteristics of microglia to investigate the potential effects of microglia in inflammatory demyelinating diseases.

Here, we present a protocol to isolate and purify primary microglia in animal models of demyelinating diseases, utilizing columnar magnetic-activated cell sorting.

Aislamiento de la microglía primaria del ratón mediante clasificación celular activada magnéticamente en modelos animales de desmielinización

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). ...

"Ordeño cerebral" de ratas para el aislamiento de células progenitoras de oligodendrocitos y tronco neural

The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new neurons and glia. One such niche is the subependymal zone (SEZ; also called the ventricular-subventricular zone), which is located across the lateral walls of the lateral ventricles, adjacent to the ependymal cell layer. Oligodendrocyte progenitor cells (OPCs) are abundantly distributed throughout the central nervous system, constituting a pool of proliferative progenitor cells that can generate oligodendrocytes.
Both NSCs and OPCs exhibit self-renewal potential and quiescence/activation cycles. Due to their location, the isolation and experimental investigation of these cells is performed postmortem. Here, we describe in detail &#34;brain milking&#34;, a method for the isolation of NSCs and OPCs, amongst other cells, from live animals. This is a two-step protocol designed for use in rodents and tested in rats. First, cells are &#34;released&#34; from the tissue via stereotaxic intracerebroventricular (i.c.v.) injection of a &#34;release cocktail&#34;. The main components are neuraminidase, which targets ependymal cells and induces ventricular wall denudation, an integrin-&#946;1-blocking antibody, and fibroblast growth factor-2. At a second &#34;collection&#34; step, liquid biopsies of cerebrospinal fluid are performed from the cisterna magna, in anesthetized rats without the need of an incision.
Results presented here show that isolated cells retain their endogenous profile and that NSCs of the SEZ preserve their quiescence. The denudation of the ependymal layer is restricted to the anatomical level of injection and the protocol (release and collection) is tolerated well&#160; by the animals. This novel approach paves the way for performing longitudinal studies of endogenous neurogenesis and gliogenesis in experimental animals.

Autor destacado: Recolección de células madre neurales y progenitoras de animales vivos utilizando un novedoso protocolo de ordeño cerebral 

A method for the isolation of neural stem cells and oligodendrocyte progenitor cells from the brains of live rats is presented here in experimental detail. It allows multiple collections of these cells from the same animals without compromising their well-being.

El método de "ordeño cerebral" para el aislamiento de células madre neurales y células progenitoras de oligodendrocitos de ratas vivas

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new ...

Isolation of Multiple Cell Types from a Mouse Brain by Mechanical Homogenization

Transcripción

Isolation of Multiple Cell Types from a Mouse Brain by Mechanical Homogenization