obtaining a single-cell suspension from mouse hippocampal tissue

Obtaining a Single-Cell Suspension from Mouse Hippocampal Tissue

Using forceps, transfer the hippocampi tissue pieces to a C-tube, and add 30 microliters of enzyme mix 2 into the tube. After twisting the cap and tightening until it clicks, place the tube in a dissociator, and run the appropriate program. While the program is running, pre-wet a 70-micron cell strainer placed on a 50-milliliter conical tube with 2 milliliters of BSA buffer.
After the dissociation program is complete, add 4 milliliters of BSA buffer to the dissociated tissue, and filter the mixture through the cell strainer on the 50-milliliter conical tube. Next, add 10 milliliters of DPBS to the C-tube. Then close the tube, swirl the solution gently, and filter it through the cell strainer on the 50-milliliter conical tube. Centrifuge the filtered cell suspension. Discard the supernatant without disturbing the pellet, and store the pellet.
For debris removal, resuspend the pellet with 1,550 microliters of cold DPBS and transfer the suspension to a labeled 15-milliliter conical tube. Then, add 450 microliters of cold debris removal solution, and pipette up and down. Gently overlay the cell suspension with 1 milliliter of cold DPBS, keeping the tip against the wall of the conical tube. Repeat the procedure until the total overlay is 2 milliliters.
Next, centrifuge the suspension at 3,000 times g for 10 minutes with full acceleration and full brake. Aspirate the topmost layer. Then, sweep the pipette tip back and forth to aspirate the white middle layer. Remove as much of the middle layer as possible without disturbing the bottom-most layer. Next, add 2 milliliters of cold DPBS, and pipette up and down to mix. Then, centrifuge the suspension at 1,000 times g for 10 minutes with full acceleration and full brake. After centrifugation, discard the supernatant, and resuspend the pellet in 1 milliliter BSA buffer.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Prepare Enzyme Mix 1 and 2 for each sample
NOTE: For volumes greater than 2 mL, use a 10 mL serologic pipette; for volumes, 200 &#181;L-2 mL, use a 1000 &#181;L pipette; for volumes, 21-199 &#181;L, use a 200 &#181;L pipette; for volumes, 2-20 &#181;L, use a 20 &#181;L pipette; for volumes under 2 &#181;L, use a 0-2 &#181;L pipette.
<ol>
	<li>For each sample, thaw one aliquot each of Enzyme P and Enzyme A at room temperature.</li>
	<li>For Enzyme mix 1, combine 50 &#181;L of Enzyme P and 1900 &#181;L of Buffer Z in a labeled C Tube (Table of Materials).</li>
	<li>For Enzyme mix 2, add 20 &#181;L of Buffer Y to the thawed 10 &#181;L aliquot of Enzyme A.</li>
</ol>
2. Adult brain dissociation protocol
NOTE: When working with samples, tubes should be placed in a tube rack at room temperature while BSA and D-PBS remain on ice unless otherwise noted.
<ol>
	<li>Switch on the dissociator.</li>
	<li>Use forceps to transfer the hippocampi tissue pieces to the C Tube.</li>
	<li>Transfer 30 &#181;L of Enzyme mix 2 into the C Tube. Twist the cap until tension is felt, then tighten until it clicks.</li>
	<li>Place the C Tube upside down into a position of the dissociator; the sample will be assigned the Selected status (Figure 1). Secure the heater over the C Tube.</li>
	<li>Press the folder icon, select Favorites folder, scroll to and select the 37C_ABDK_02 program. Click on OK to apply the program to all selected C tubes, then tap on Start (Figure 1).</li>
	<li>Label one 50 mL conical tube per sample.</li>
	<li>Place a 70 &#181;m cell strainer on each 50 mL conical tube and wet with 2 mL of BSA buffer.</li>
	<li>Upon completion of the program, remove the heater and the C tube from the dissociator.</li>
	<li>Add 4 mL of BSA buffer to the sample and apply the mixture to the cell strainer on the 50 mL conical tube.</li>
	<li>Add 10 mL of D-PBS to the C Tube, close it, and swirl the solution gently. Apply it to the cell strainer on the 50 mL conical tube.</li>
	<li>Discard the cell strainer and the C Tube. Centrifuge the suspension at 300 x g for 10 min at 4&#176;C. Then, aspirate and discard the supernatant.</li>
</ol>
3. Debris removal
<ol>
	<li>Resuspend the pellet with 1550 &#181;L of cold D-PBS and transfer the suspension to a labeled 15 mL conical tube.</li>
	<li>Add 450 &#181;L of cold Debris Removal Solution and pipette up and down (do not vortex). 
	NOTE: Debris Removal Solution is a reagent in the commercial Adult Brian Dissociation Kit.</li>
	<li>Gently overlay 1 mL of cold D-PBS on top of the cell suspension, keeping the tip against the wall of the conical tube. Repeat until the total overlay is 2 mL. 
	NOTE: This step is critical; perform with proficiency and dexterity.</li>
	<li>Centrifuge at 3000 x g for 10 min at 4&#176;C with full acceleration and full brake. 
	NOTE: If the phases are not clearly separated, repeat steps 7.2-7.3. Centrifuge a final time at 1000 x g for 10 min at 4&#176;C.</li>
	<li>The suspension should now consist of three distinct layers (Figure 2). Aspirate the topmost layer. Sweep the pipette tip back and forth to aspirate the white middle layer. Remove as much of the middle layer as possible without disturbing the bottom-most layer. 
	NOTE: This step is critical; perform with proficiency and dexterity.</li>
	<li>Add 2 mL of cold D-PBS and pipette up and down to mix.</li>
	<li>Centrifuge at 1000 x g for 10 min at 4&#176;C with full acceleration and full brake. Aspirate and discard the supernatant. Resuspend the pellet in 1 mL of BSA buffer. 
	&#8203;NOTE: Cells can be resuspended in the appropriate buffer then magnetically labeled and isolated in preparation for single-cell sequencing at this point.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Microcentrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>02-682-003</td>
			<td>Basix, assorted color</td>
		</tr>
		<tr>
			<td>15 mL Falcon Tubes</td>
			<td>Becton Dickinson Labware Europe</td>
			<td>352009</td>
			<td>Polystyrene</td>
		</tr>
		<tr>
			<td>25 mL Serological Pipets</td>
			<td>Fisher Scientific</td>
			<td>14-955-235</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainer</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Adult Brain Dissociation Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-107-677</td>
			<td>Contains Enzyme P, Buffer Z, Buffer Y, Enzyme A, Buffer A, Debris Removal Solution</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS C Tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Octo Dissociator with Heaters</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-427</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco DPBS (1X)</td>
			<td>ThermoFisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Beaker</td>
			<td>Fisher Scientific</td>
			<td>02-555-25A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips GP LTS 20 &#181;L 960A/10</td>
			<td>Rainin</td>
			<td>30389270</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips GP LTS 250 &#181;L 960A/10</td>
			<td>Rainin</td>
			<td>30389277</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips RT LTS 1000 &#181;L FL 768A/8</td>
			<td>Rainin</td>
			<td>30389213</td>
			<td></td>
		</tr>
		<tr>
			<td>Rainin Pipet-Lite XLS (2, 20, 200, 1000 &#956;L)</td>
			<td>Rainin</td>
			<td>30386597</td>
			<td></td>
		</tr>
		<tr>
			<td>Round Ice Bucket with Lid</td>
			<td>Fisher Scientific</td>
			<td>07-210-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-Bottom Tubes with Cell Strainer Cap</td>
			<td>Falcon</td>
			<td>100-0087</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Trujillo, M., et al. <a href="https://app.jove.com/v/63007">Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue.</a> J. Vis. Exp. (2021)
This video demonstrates a protocol to obtain a highly viable single-cell suspension from the mouse brain hippocampus using a combination of automated mechanical dissociation and a mixture of enzymes to aid in enzymatic digestion.

<img alt="Figure 1" src="/files/ftp_upload/22588/22588_Figure1.jpg" /> 
Figure 1: Selected status in step 2.4
<img alt="Figure 2" src="/files/ftp_upload/22588/22588_Figure2.jpg" /> 
Figure 2: Tri-layered suspension in step 3.5: Buffer (top layer), cell debris, debris removal solution, and cells (bottom layer).

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Prepare Enzyme Mix 1 and 2 ...

Take perfused adult mouse hippocampi fragments in a dissociation tube containing papain and DNase.
Place the tube on a mechanical dissociator.
The dissociator mechanically disintegrates the tissue, loosening cells, including neurons and glial cells.
Papain breaks down the tissue&#39;s extracellular matrix, releasing cells. DNase degrades contaminating DNA.
Add buffer containing BSA, inactivating the enzymes.
Pass the suspension through a strainer to remove large debris.
Centrifuge the filtrate. Discard the enzyme-rich supernatant.
Resuspend the cells in buffer, and transfer them to a fresh tube.
Add density gradient media, mix, and overlay the mixture with buffer.
During centrifugation, cells and debris form separate layers based on their densities.
Remove the topmost buffer layer and the middle debris layer.&#160;
Add buffer to the bottom layer containing the cells and mix. Centrifuge and discard the supernatant containing the density gradient media.
Resuspend the hippocampal cells in buffer for further analysis.

26 Views. Uzyskiwanie zawiesiny jednokomórkowej z tkanki hipokampa myszy

Video: Uzyskiwanie zawiesiny jednokomórkowej z tkanki hipokampa myszy - Experiment

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA sequencing and primary cell culture). Yielding viable, healthy cells in large quantities is critical, and the optimal conditions to do so are tissue dependent. Cell populations in the tongue epithelium and underlying mesenchyme/connective tissue are heterogeneous and tissue structures vary in different regions and at different developmental stages. We have tested protocols for isolating cells from the mouse tongue epithelium and mesenchyme/connective tissue in the early developmental [embryonic day 12.5 (E12.5)] and young adult (8-week) stages. A clean separation between the epithelium and underlying mesenchyme/connective tissue was easy to accomplish. However, to further process and isolate cells, yielding viable healthy cells in large quantities, and careful selection of enzymatic digestion buffer, incubation time, and centrifugation speed and time are critical. Incubation of separated epithelium or underlying mesenchyme/connective tissue in 0.25% Trypsin-EDTA for 30 min at 37 &#176;C, followed by centrifugation at 200 x g for 8 min resulted in a high yield of cells at a high viability rate (&#62;90%) regardless of the mouse stages and tongue regions. Moreover, we found that both dissociated epithelial and mesenchymal/connective tissue cells from embryonic and adult tongues could survive in the cell culture-based medium for at least 3 h without a significant decrease of cell viability. The protocols will be useful for studies that require the preparation of isolated cells from mouse tongues at early developmental (E12.5) and young adult (8-week) stages requiring cell dissociation from different tissue compartments.

We have developed a generalized protocol to dissociate a large quantity of high-quality single cells from the epithelium and mesenchyme/connective tissue of embryonic and adult mouse tongues.

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA ...

JoVE Journal

Developmental Biology

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal neurons allows the study of individual neurons or subcellular components. We have shown subcellular protein localization within a neuron by immunocytochemistry, neuronal polarity, synaptic morphology, and its developmental change using a low-density primary hippocampal culture. Recently, ready-to-use frozen stocks of neurons have become commercially available. These frozen stocks of neurons reduce the time needed to prepare animal experiments and also contribute to the reduction of the number of animals used. Here, we introduce a reproducible low-density primary culture method using a 96-well plate. We used a commercially available frozen stock of neurons from the rat embryonic hippocampus. The neurons can be stably cultured long-term without media changes by reducing the growth of glial cells at particular timepoints. This high-throughput assay using low-density culture allows reproducible imaging-based evaluations of synaptic plasticity.

A ready-to-use frozen stock of neurons is a powerful tool for evaluating synaptic functions. Here, we introduce an easy low-density primary culture from frozen stock using a 96-well plate.

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal ...

Obtaining a Single-Cell Suspension from Mouse Hippocampal Tissue

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Obtaining a Single-Cell Suspension from Mouse Hippocampal Tissue