obtaining various neural cell populations from a rat hippocampus

Obtaining Various Neural Cell Populations from a Rat Hippocampus

Begin the neural dissociation by using sterile razor blades to dice each sample harvested from adult brain tissues into small 30 to 400-milligram pieces on the lid of a six-well culture plate. Then immerse the tissue in 1 milliliter of calcium- and magnesium-free HBSS, and use a pipette to transfer the samples into sterile 2-milliliter centrifuge tubes.
When all of the tissues have been collected, centrifuge the samples, and aspirate the supernatants, and incubate the samples for 15 minutes in a 37 degrees Celsius water bath, inverting the tube several times every five minutes to resuspend the settled pieces of tissue.
At the end of the incubation, add 30 microliters of freshly prepared enzyme mixed tube to each sample, and gently invert the tubes. Then, using a Pasteur pipette labeled 1, dissociate each sample with 30 up and down trituration, taking care to avoid generating bubbles. Incubate the samples for another 15 minutes at 37 degrees Celsius with inversion every five minutes, followed by dissociation of the samples with Pasteur pipette number 2, as just demonstrated.
Then, switch to pipette number 3. Repeat the dissociation and incubate the samples for 10 more minutes in the water bath, with inversion every five minutes. At the end of the incubation, filter the single-cell suspensions through 80-micron cell strainers into 15-milliliter Falcon tubes, washing each strainer with 10 milliliters of calcium- and magnesium-free HBSS to stop the enzymatic reactions. When the last sample has been filtered, spin down the tubes, and aspirate the supernatants.
To deplete the myelin from the cell suspensions, thoroughly resuspend the pellets in 400 microliters of myelin removal buffer, and add the appropriate amount of myelin removal beads to each tube. Pipette the solutions up and down until the cells and beads are thoroughly mixed. Then, incubate the sample at 4 degrees Celsius for 15 minutes. After the incubation, wash each sample in 5 milliliters of myelin removal buffer.
While the cells are spinning, place one column for each sample in the magnetic field of a magnetic sorter, and place a clean 80-micron filter on top of each column. Rinse each filter and column with 1 milliliter of myelin removal buffer three times, collecting all of the flow-through in a waste container.
After the final rinse, place 5-milliliter polystyrene round-bottom tubes directly beneath each column to collect the eluate. Then, aspirate the supernatant from the samples, and resuspend the pellets in 500 microliters of myelin removal buffer.
Immediately apply the cell suspensions to the columns, collecting the cells in the tubes below. Then, rinse each filter and column 4 times with 1 milliliter of myelin removal buffer per wash. After the last wash, spin down the cells and aspirate the supernatants.

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Neuronal Culture Techniques

Primary Neuronal Culture

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Tissue Collection
<ol>
	<li>Prepare Buffer X from the Neural Dissociation Kit by adding beta-mercaptoethanol to Buffer X to a final concentration of 0.067 mM (for 4 samples, add 13.5 &#181;l of 50 mM beta-mercaptoethanol to 10 ml of Buffer X).</li>
	<li>Prepare Enzyme Mix 1 by mixing 50 &#181;l of Enzyme P with 1,900 &#181;l of Buffer X for each sample as described in the protocol for the Neural Dissociation Kit For Example, for 4 samples, mix 200 &#181;l of Enzyme P with 7,600 &#181;l of Buffer X.</li>
	<li>Place Buffer X in the water bath at 37 &#186;C.</li>
	<li>Place 2 ml of cold Hank&#39;s Buffered Salt Solution (HBSS) (without Ca++ and Mg++) into one well of a sterile 6-well culture plate for each sample that will be collected and kept on ice for tissue dissection.</li>
</ol>
2. Neural Dissociation (1 hr)
<ol>
	<li>Dice each tissue sample into small pieces using sterile razor blades on the lid of the 6-well culture plate. This protocol has been used for tissue samples of approximately 30 - 100 mg, but is approved for tissue samples of up to 400 mg. For tissue samples larger than 400 mg, the reagent volumes must be scaled up appropriately.</li>
	<li>Use 1 ml of fresh HBSS without Ca2+ and Mg2+ to transfer the diced pieces of tissue with a pipette into a sterile 2 ml centrifuge tube. Repeat steps 2.1 and 2.2 for all sample until all samples have been transferred into a 2 ml centrifuge tube.</li>
	<li>Centrifuge the samples at 300 x g for 2 min at RT and aspirate supernatant.</li>
	<li>Add 1,900 &#181;l of warm Enzyme Mix 1, as prepared in Steps 1.2 and 1.3, to each sample and close the tubes.</li>
	<li>Incubate the samples for 15 min in the 37 &#186;C water bath, inverting the tubes several times every 5 min to re-suspend the settled pieces of tissue.</li>
	<li>Meanwhile, prepare Enzyme Mix 2 by mixing 20 &#181;l of Buffer Y with 10 &#181;l of thawed Enzyme A (for 4 samples, mix 80 &#181;l of Buffer Y with 40 &#181;l of thawed Enzyme A).</li>
	<li>Add 30 &#181;l of Enzyme Mix 2 to each sample and invert gently. Do not vortex.</li>
	<li>Dissociate each tissue sample with Pasteur pipette &#34;1&#34;, triturating up and down 30 times.</li>
	<li>Incubate the samples for 15 min in the 37 &#186;C water bath; invert the tubes several times every 5 min to re-suspend the settled pieces of tissue.</li>
	<li>Dissociate each tissue sample with Pasteur pipette &#34;2,&#34; triturating up and down 30 times, avoiding generating bubbles.</li>
	<li>Dissociate each tissue sample with Pasteur pipette &#34;3,&#34; triturating up and down 30 times, avoiding generating bubbles.</li>
	<li>Incubate the samples for 10 min in the 37 &#186;C water bath; invert the tubes several times every 5 min to re-suspend the settled cells.</li>
	<li>Apply single-cell suspension to an 80-micron cell strain placed on top of a 15 ml falcon tube to remove any large pieces of debris and wash the filter and cells with 10 ml of HBSS (with Ca2+ and Mg2+) to stop the enzyme reactions. Repeat until each sample is transferred into a clean 15 ml falcon tube.</li>
	<li>Centrifuge the samples at 300 x g for 10 min at RT and aspirate off the supernatant.</li>
</ol>
3. Myelin Depletion (45 min)
<ol>
	<li>Thoroughly re-suspend the pellet of cells with 400 &#181;l of myelin removal buffer.</li>
	<li>Add a specified amount of the myelin removal beads and pipette up and down to thoroughly mix. 
	NOTE: The volume of myelin removal beads used will depend upon the size of the tissue, the age of the animal, and the amount of myelin expected in that particular brain region. For example, for an adult rat hippocampus sample (approximately 300 mg), incubate the sample with 100 &#181;l of myelin removal beads.</li>
	<li>Incubate for 15 min in the refrigerator at 4 &#186;C.</li>
	<li>Wash each sample with 5 ml of myelin removal buffer.</li>
	<li>Centrifuge the samples at 300 x g for 10 min at RT.</li>
	<li>While centrifuging the samples, place 1 column for each sample in the magnetic field of the magnetic sorter and place a clean 80-micron filter on top of each column.</li>
	<li>Prepare the columns and filter by rinsing each column with 1 ml of myelin removal buffer three times (3 ml total). Collect all flow-through in a waste container, such as the bottom of an empty tip box.</li>
	<li>Position a 5 ml polystyrene round bottom tube directly underneath each column in preparation of sample collection.</li>
	<li>When the cells have finished centrifugation, aspirate the supernatant and thoroughly re-suspend it in 500 &#181;l of myelin removal buffer. Immediately apply the cell suspension to the column and collect the cells in the tube below.</li>
	<li>Wash the filter and column with 1 ml myelin removal buffer, 4 times (4 ml total).</li>
	<li>Centrifuge the cell collection at 300 x g for 10 min at RT.</li>
	<li>Aspirate the supernatant and the cells are now ready for staining.</li>
</ol>
4. Staining Live Cells for FACS (1 hr for staining)
<ol>
	<li>Briefly vortex (2 sec) the cells to separate the pellet and immediately add 5 &#181;l of Fc block (anti-clusters of differentiation 32, CD32), vortex again, and incubate in the refrigerator at 4 &#186;C for 5 min.</li>
	<li>Meanwhile, prepare the primary antibody mixture by adding the three primary antibodies in wash buffer as follows: 0.125 &#181;l of allophycocyanin (APC) -conjugated CD11b antibody (1:800), 1 &#181;l of anti-GLT1 antibody (1:100), and 0.4 &#181;l of Fluorescein isothiocyanate (FITC) &#8211; conjugated Thy1 antibody (1:250) for every 100 &#181;l of wash buffer. Each sample will receive 100 &#181;l of primary antibody mixture for incubation; however, if your sample is much larger than 400 mg of tissue, you may want to consider increasing the volume of your primary antibody mixture accordingly</li>
	<li>Prepare tubes for single staining controls by removing just 2 &#181;l of cells from each sample tube and adding it to each single stain control tube. The &#34;single stain&#34; controls include: APC-CD11b only, GLT1 only, FITC-Thy1 only, PE secondary antibody only, and the &#34;blank&#34; or unstained control. 
	NOTE: All samples should be represented in all staining controls, thus 2 &#181;l of cells from each sample should be added to each staining control tube.</li>
	<li>Briefly vortex the tubes (2 sec) and add 100 &#181;l of primary antibody mixture to each sample tube and 100 &#181;l of appropriate primary antibody to each staining control tube, vortex the tubes again, cover the tubes with aluminum foil to protect the fluorescent antibodies from any light, and incubate the tubes in the refrigerator at 4 &#186;C for 20 min. The samples should be kept cool (at 4 &#186;C) for the remainder of the procedure to maintain the integrity of the samples and the antibodies.</li>
	<li>Briefly vortex the tubes and add 2 ml of wash buffer to each tube.</li>
	<li>Centrifuge tubes at 350 x g for 5 min at 4 &#186;C.</li>
	<li>Meanwhile, prepare the secondary antibody mixture by adding 0.2 &#181;l of PE-conjugated anti-rabbit secondary antibody for every 100 &#181;l of wash buffer. Each sample will get 100 &#181;l of secondary antibody mixture; however, if your sample is much larger than 400 mg of tissue, you may want to consider increasing the volume of your primary antibody mixture accordingly.</li>
	<li>When the centrifugation is complete, dump the supernatant off of each sample into the sink and blot the tubes upside-down on a paper towel.</li>
	<li>Briefly vortex the tubes and add 100 &#181;l of secondary antibody mixture to each sample tube and the appropriate secondary antibody to each staining control. Then, vortex the tubes again, cover them with aluminum foil, and incubate them in the refrigerator at 4&#186;C for 15 min. 
	NOTE: The GLT1-only staining control tube should get the secondary antibody mixture.</li>
	<li>After the incubation, briefly vortex the tubes and add 2 ml of wash buffer to each tube.</li>
	<li>Centrifuge the tubes at 350 x g for 5 min at 4&#186;C.</li>
	<li>When the centrifugation is complete, dump the supernatant off of each sample into the sink and blot the tubes upside-down on a paper towel.</li>
	<li>Vortex the tubes and add 0.25 ml of sterile PBS to each tube. The samples are ready for sorting. 
	NOTE: More cells may require 0.5 ml of sterile PBS; however it is best to begin with a lower volume as the sample can always be diluted if it is too concentrated with cells. 
	NOTE: Optionally add DNase to the tube to prevent the cells from clumping and thus clogging the sorter. Samples can also be filtered using a sterile 80 micron filter just prior to sorting to avoid clogging the sorter.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Neural Dissociation Kit (P)</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-628</td>
			<td></td>
		</tr>
		<tr>
			<td>Myelin Removal Beads II</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-733</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>QuadroMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Mesh Sheet</td>
			<td>Amazon</td>
			<td>CMN-0074-10YD</td>
			<td>40 inch width, 80 micron size mesh</td>
		</tr>
		<tr>
			<td>Fc Block / anti-CD32</td>
			<td>BD Biosciences</td>
			<td>BDB550270</td>
			<td>Reactivity for rat</td>
		</tr>
		<tr>
			<td>APC-conjugated CD11b antibody</td>
			<td>Biolegend</td>
			<td>201809</td>
			<td>Reactivity for rat</td>
		</tr>
		<tr>
			<td>Rabbit anti-GLT1</td>
			<td>Novus Biologicals</td>
			<td>NBP1-20136</td>
			<td>Reactivity for rat or human</td>
		</tr>
		<tr>
			<td>PE-conjugated anti-rabbit secondary antibody</td>
			<td>eBioscience</td>
			<td>1037259</td>
			<td>Secondary antibody for anti-GLT1</td>
		</tr>
		<tr>
			<td>FITC-conjugated anti-rat CD90 (Thy1) mouse antibody</td>
			<td>Biolegend</td>
			<td>202504</td>
			<td>Reactivity for rat</td>
		</tr>
	</tbody>
</table>

Source: Schwarz, J. M. <a href="https://app.jove.com/v/52537">Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue.</a> J. Vis. Exp. (2015)
This video demonstrates a protocol to isolate cells from rat hippocampi, including neurons, astrocytes, and microglia.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Tissue Collection

	Prepare ...

Take perfused rat hippocampus fragments.
Incubate with papain to digest the tissue&#39;s extracellular matrix and loosen cells, including neurons, astrocytes, and microglia.
Add DNase and mix.
Mechanically dissociate the tissue using pipettes of progressively decreasing diameters to obtain a single-cell suspension. Incubate for the DNase to degrade contaminating DNA.
Pass the suspension through a strainer, removing debris.
Add buffer containing divalent ions to stop the enzymatic activity.
Centrifuge and remove the enzyme-rich supernatant.
Now, add buffer and anti-myelin magnetic beads. Incubate for the beads to bind the myelin debris.
After incubation, centrifuge and remove the supernatant.
Resuspend the mixture in buffer and load it onto a magnetic separation column containing ferromagnetic spheres placed in the magnetic field of the magnetic sorter.
The magnetic field retains magnetic bead-bound myelin debris in the column while myelin-free cells pass through.
Wash the column with buffer and collect the flow-through containing neurons, astrocytes, and microglia.

31 visualizações. Obtenção de várias populações de células neurais de um hipocampo de rato

Vídeo: Obtenção de várias populações de células neurais de um hipocampo de rato - Experimento

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

Fluorescência de células activadas (FACS) e Gene Análise da expressão de Fos-expressando neurônios de fresco e congelado cérebro de um rato Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

JoVE Journal

Neurociência

Fluorescence-Activated Nuclei Negative Sorting of Neurons Combined with Single Nuclei RNA Sequencing to Study the Hippocampal Neurogenic Niche

Adult Hippocampal Neurogenesis (AHN), which consists of a lifelong maintenance of proliferative and quiescent neural stem cells (NSCs) within the sub-granular zone (SGZ) of the dentate gyrus (DG) and their differentiation from newly born neurons into granule cells in the granule cell layer, is well validated across numerous studies. Using genetically modified animals, particularly rodents, is a valuable tool to investigate signaling pathways regulating AHN and to study the role of each cell type that compose the hippocampal neurogenic niche. To address the latter, methods combining single nuclei isolation with next generation sequencing have had a significant impact in the field of AHN to identify gene signatures for each cell population. Further refinement of these techniques is however needed to phenotypically profile rarer cell populations within the DG. Here, we present a method that utilizes Fluorescence Activated Nuclei Sorting (FANS) to exclude most neuronal populations from a single nuclei suspension isolated from freshly dissected DG, by selecting unstained nuclei for the NeuN antigen, in order to perform single nuclei RNA sequencing (snRNA-seq). This method is a potential steppingstone to further investigate intercellular regulation of the AHN and to uncover novel cellular markers and mechanisms across species.

Presented here is a method to sequence single nuclei isolated from the mouse dentate gyrus that excludes most neurons through fluorescence-activated nuclei (FAN)-sorting. This approach generates high-quality expression profiles and facilitates the study of most other cell types represented in the niche, including scarce populations such as neural stem cells.

Classificação negativa de neurônios por núcleos ativados por fluorescência combinada com sequenciamento de RNA de núcleos únicos para estudar o nicho neurogênico do hipocampo

Adult Hippocampal Neurogenesis (AHN), which consists of a lifelong maintenance of proliferative and quiescent neural stem cells (NSCs) within the ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

Classificação celular ativada pela fluorescência para o isolamento de populações celulares escleracianas

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

Obtaining Various Neural Cell Populations from a Rat Hippocampus

Transcrição

Obtaining Various Neural Cell Populations from a Rat Hippocampus