a technique to isolate nuclei from non-neuronal cells in the hippocampal dentate gyrus

A Technique to Isolate Nuclei from Non-neuronal Cells in the Hippocampal Dentate Gyrus

Homogenize the tissue with 10 strokes of the loose A pestle, followed by 15 strokes of the tight B pestle. Transfer the homogenate into a pre-chilled 15-milliliter tube. Rinse the Dounce homogenizer with 1 milliliter of cold HB, and combine it with the same tube.
Add 3 milliliters of HB to the 15-milliliter tube, and incubate for 5 minutes on ice. Mix this nuclei suspension twice by inverting the tube gently. Using 0.5 milliliters of HB, pre-wet the 70-micrometer strainer cap placed over a 50-milliliter test tube. Then, strain the nuclei suspension before washing the cell strainer with 0.5 milliliters of HB.
Next, remove the cell strainer, and centrifuge the test tube in a swing bucket centrifuge at 500 g for 5 minutes at 4 degrees Celsius. Discard the supernatant. Gently resuspend the pellet in 4 milliliters of HB using a P1000 pipette.
After five minutes of incubation on ice, centrifuge the suspension at 500 g for 10 minutes at 4 degrees Celsius. Discard the supernatant, and resuspend the pellet in 3 milliliters of wash media. Use 0.5 milliliters of wash media to pre-wet a 35-micrometer strainer cap over a 50-milliliter test tube. Then, strain the nuclei suspension by pipetting 0.5 milliliters of suspension at a time using P1000 pipette.
After washing the strainer cap with 0.5 milliliters of wash media, place the tube on ice. Transfer the filtrate to a new 15-milliliter tube, and centrifuge it for 5 minutes and 4 degrees Celsius at 500 g. Discard the supernatant, and resuspend the pellet in 3 milliliters of wash media.
Repeat the centrifugation, and resuspend the pellet in 1 milliliter of wash media with the mouse anti-Neu and AlexaFlour488-conjugated antibody and 1 microgram per milliliter DAPI. Incubate the reaction for 45 minutes on ice in the dark.
For Fluorescence Activated Nuclei Sorting or FANS, transfer the immunostained nuclei suspension to a 5-milliliter test tube, and place it on ice until the start of the flow cytometry procedure.
Vortex the samples for three seconds with mild intensity before placing the tubes into the FACS instrument.
To acquire the data from stain nuclei suspension, set the gates in DAPI height and the DAPI area to exclude the cell debris and the aggregated nuclei. Then, set the gates in the log side-scatter or SSC area and the log forward-scatter or FSC area to separate the single nuclei from the remaining DAPI-stained aggregate or cell debris.
Then, isolate the NeuNAF488-negative population by setting the gates for the anti-NeuNAF488 and FSC area.
After the analysis, sort the anti-NeuNAF488-negative population in a 1.5-milliliter collection tube filled with 50 microliters of wash media using the gating strategy.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines
1. Dissection of the DG (Timing: 15 min)
<ol>
	<li>Prepare nuclei isolation media 1 and 2 (NIM1 and NIM2), homogenization buffer (HB), and wash media (WM) (Table 1). Place all the buffers, media, reagents, and tools on ice until needed. Put the Dounce homogenizer (see Table of Materials) on ice during preparation (minimum 1 h before the homogenization step). 
	NOTE: NIM1 can be prepared and stored at 4 &#176;C for up to 6 months. NIM2, HB, and WM should be freshly prepared. 
	CAUTION: Manipulate dithiothreitol (DTT), the protease inhibitor and Triton X-100 with care. These compounds are skin and eye irritating, acutely toxic, and hazardous for the aquatic environment. While using these chemicals wear protective gloves, clothing, eye and face protection, wash hands thoroughly after handling, and avoid release to the environment.</li>
	<li>Euthanize a 22-month-old male C57Bl/6J mouse by cervical dislocation following the Home Office Schedule 1 procedure. 
	NOTE: See discussion for rationale regarding the use of 22-month-old mouse in this study. However, this protocol can be performed at any age across the lifespan.</li>
	<li>Dissect the brain out from an euthanized mouse and transfer it into a 10 cm Petri dish filled with ice-cold 1x phosphate-buffered saline (PBS) (Figure 1). Place the Petri dish on ice. Remove the cerebellum using a scalpel and cut the brain in half between both the hemispheres (along the sagittal axis).</li>
	<li>Fill in a new 10 cm Petri dish with ice-cold PBS and place it on ice. Transfer one half of the brain to the new Petri dish. Using binoculars, dissect out the DG and repeat this step to obtain the second DG from the second half of the brain. 
	&#8203;NOTE: These steps (steps 1.2-1.4) were adapted from a previously described procedure. It is important to proceed as rapidly as possible at this stage to preserve the integrity of the cells.</li>
	<li>Transfer the two DGs into the precooled Dounce homogenizer and add 1 mL of cold HB.</li>
</ol>
2. Tissue dissociation, single nuclei isolation, and anti-NeuN immunostaining (Timing: 2 h)
<ol>
	<li>Homogenize the tissue with 10 strokes of the loose &#34;A&#34; pestle, followed by 15 strokes of the tight &#34;B&#34; pestle. 
	NOTE: Dounce homogenization should be performed with the mortar on ice with gentle strokes to reduce heat caused by friction and foaming. All the buffers and equipment should be precooled and kept on ice during the procedure.</li>
	<li>Transfer the homogenate into a prechilled 15 mL tube; rinse the Dounce homogenizer with 1 mL of cold HB and combine to the same tube. Add 3 mL of HB to the 15 mL tube and incubate 5 min on ice. Mix 2x by inverting the tube gently.</li>
	<li>Pre-wet a 70 &#181;m strainer cap with 0.5 mL of HB over a 50 mL test tube. Strain the nuclei suspension from step 2.2 by tipping over the 15 mL tube gently into the cell strainer. Wash the cell strainer with 0.5 mL of HB.</li>
	<li>Remove the cell strainer and centrifuge the test tube at 500 x g for 5 min at 4 &#176;C, using a swing bucket centrifuge. Discard the supernatant. 
	NOTE: Straining the homogenate will help reduce the debris, which is critical for the flow cytometry and the single nuclei RNA sequencing (snRNA-seq) downstream steps.</li>
	<li>Resuspend the pellet gently in 4 mL of HB using a P1000 pipette. Incubate on ice for 5 min. Spin at 500 x g for 10 min at 4 &#176;C. Discard the supernatant and resuspend the pellet in 3 mL of WM.</li>
	<li>Pre-wet a 35 &#181;m strainer cap over a 15 mL test tube with 0.5 mL of WM. Strain the nuclei suspension from step 2.5 through the cell strainer, gently pipetting 0.5 mL at a time using a P1000 pipette.</li>
	<li>Wash the strainer cap with 0.5 mL of WM and place the tube on ice. Transfer the filtrate to a new 15 mL tube and centrifuge for 5 min and 4 &#176;C at 500 x g. Discard the supernatant and resuspend the pellet in 3 mL of WM.</li>
	<li>Spin at 500 x g for 5 min at 4 &#176;C. Discard the supernatant and resuspend the pellet in 1 mL of WM with the mouse anti-neuronal nuclear antigen (NeuN), AlexaFluor 488-conjugated antibody (anti-NeuN-AF488, 1:32,000) and 1 &#181;g/mL 4&#8242;,6-diamidino-2-phenylindole (DAPI). Incubate for 45 min on ice in the dark. 
	NOTE: To optimize the immunostaining of isolated nuclei, it is recommended to titrate the antibody to determine the optimal dilution for flow cytometry analysis and sorting. Then, run adequate controls to confirm that the staining conditions are optimal. For instance, with the conjugated anti-NeuN-AF488 antibody, a negative control (i.e., no addition of the antibody, Figure 2A) and a positive control (i.e., staining with the antibody, Figure 2B) were run to assess segregation of unstained and stained populations. When starting to work with an AF488-conjugated antibody, running an AF488-conjugated isotype control is recommended to assess specificity. If a non-conjugated antibody is used, additional control such as adding a secondary antibody only to a nuclei preparation might be necessary to assess unspecific binding of the secondary antibody.</li>
</ol>
3. Fluorescence-activated nuclei sorting (FANS) to exclude neuronal populations (Timing: 45 min)
<ol>
	<li>Transfer the immuno-stained nuclei suspension to a 5 mL test tube and keep it on ice until the start of the flow cytometry procedure. 
	NOTE: If working with bigger pieces of tissue than two mouse DG, further dilution with WM buffer might be required to avoid clogging the fluorescence-activated cell sorter (FACS) if nuclei density in the solution becomes high.</li>
	<li>Vortex the samples for 3 s at low speed before placing the tubes into the FACS instrument (see Table of Materials). 
	NOTE: (FACS setup) Sorting machines need to be aligned at the start of the procedure with calibration particles following the manufacturer&#39;s recommendations. The drop delay was calibrated with beads or microspheres (see Table of Materials) according to the FACS model. Samples were sorted at 4 &#176;C on purity mode. To reduce the collection volume, the nuclei were sorted through a 70 &#181;m nozzle at the recommended pressure for the flow cytometer. Nuclei were sorted into 1.5 mL low binding tubes (see Table of Materials) containing 50 &#181;L of WM. All collection tubes were coated in PBS + 5% bovine serum albumin (BSA) at 4 &#176;C overnight to reduce the risk of nuclei sticking to the walls of the tube.</li>
	<li>To acquire the data from a sample of the stained nuclei suspension, set the gates in DAPI-height and the DAPI-area in order to exclude the cell debris and the aggregated nuclei (Figure 3A). Furthermore, separate single nuclei from any remaining DAPI-stained aggregates or cell debris by setting the gates in the log side scatter (SSC)-area and the log forward scatter (FSC)-area (Figure 3B).</li>
	<li>Set the gates for the anti-NeuN-AF488 and the FSC-area, to isolate the NeuN-AF488-negative population, as shown in Figure 3C.</li>
	<li>After analysis, using the gating strategy described above, sort the NeuN-AF488-negative population in a 1.5 mL collection tube filled with 50 &#181;L of WM. 
	NOTE: Following the gating strategy described above and the dissection procedure to isolate the DG from the brain of an adult mouse, the NeuN-AF488-negative population is expected to represent ~14% of the single nuclei.</li>
</ol>
Table 1: Compositions of media and buffers used in the study.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Nuclei isolation medium #1 (NIM1) &#8211; store up to 6 months at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Volume (&#181;L)</td>
			<td>Final concentration (mM)</td>
		</tr>
		<tr>
			<td>1.5 M Sucrose</td>
			<td>1,833.30</td>
			<td>250</td>
		</tr>
		<tr>
			<td>1 M KCl</td>
			<td>275</td>
			<td>25</td>
		</tr>
		<tr>
			<td>1 M MgCl2</td>
			<td>55</td>
			<td>5</td>
		</tr>
		<tr>
			<td>1 M Tris buffer, pH 8.0</td>
			<td>110</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>8,726.70</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>11,000</td>
			<td>-</td>
		</tr>
		<tr>
			<td colspan="3">Nuclei isolation medium #2 (NIM2) &#8211; make fresh</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Volume (&#181;L)</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>NIM1</td>
			<td>10,769</td>
			<td>-</td>
		</tr>
		<tr>
			<td>1 mM DTT</td>
			<td>11</td>
			<td>1 &#181;M</td>
		</tr>
		<tr>
			<td>100x Protease inhibitor</td>
			<td>110</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Triton X-100 10% (v/v)</td>
			<td>110</td>
			<td>0.1% (v/v)</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>11,000</td>
			<td>-</td>
		</tr>
		<tr>
			<td colspan="3">Homogenization buffer (HB) &#8211; make fresh</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Volume (&#181;L)</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>NIM2</td>
			<td>10,958.75</td>
			<td>-</td>
		</tr>
		<tr>
			<td>RNase Inhibitor, 40 U &#181;L-1</td>
			<td>27.5</td>
			<td>100 U mL-1</td>
		</tr>
		<tr>
			<td>RNasin, 40 U &#181;L-1</td>
			<td>13.75</td>
			<td>50 U mL-1</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>11,000</td>
			<td>-</td>
		</tr>
		<tr>
			<td colspan="3">Wash Media (WM)</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Volume (&#181;L)</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>BSA 7.5%</td>
			<td>1,466.70</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>RNase Inhibitor, 40 U &#181;L-1</td>
			<td>27.5</td>
			<td>100 U mL-1</td>
		</tr>
		<tr>
			<td>RNasin, 40 U &#181;L-1</td>
			<td>13.75</td>
			<td>50 U mL-1</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>9,492.05</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>11,000</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5ml microtube</td>
			<td>Eppendorf</td>
			<td>30124537</td>
			<td></td>
		</tr>
		<tr>
			<td>10.00&#181;m Flouresbrite YG Carboxylate Microspheres</td>
			<td>Polysciences</td>
			<td>15700-10</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL polypropylene centrifuge tubes</td>
			<td>Corning</td>
			<td>430052</td>
			<td></td>
		</tr>
		<tr>
			<td>2 pairs of sterile Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-30</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Sigma Aldrich</td>
			<td>D9564-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>4150 TapeStation System</td>
			<td>Agilent</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL round bottom high clarity polypropylene test tube with snap cap</td>
			<td>Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL round bottom polystyrene test tube with cell strainer snap cap</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL polypropylene centrifuge tubes</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>8 peak SPHERO Rainbow Calibration Particles</td>
			<td>BD Biosciences</td>
			<td>RCP-30-5A</td>
			<td></td>
		</tr>
		<tr>
			<td>Accudrop Beads</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra X-30R Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-NeuN antibody, clone A60, Alexa Fluor 488 conjugated</td>
			<td>Millipore</td>
			<td>MAB377X</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;BD FACSAria Fusion Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Coulter MoFlo XDP</td>
			<td>Beckman Coulter</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA 7.5%</td>
			<td>Gibco</td>
			<td>15260037</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Thermo Scientific</td>
			<td>R0861</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce tissue grinder set: mortar, loose pestle (A) and tight pestle (B)</td>
			<td>KIMBLE</td>
			<td>D8938-1SET</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Tubes Protein LoBind 1.5ml</td>
			<td>Eppendorf</td>
			<td>30108116</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Halt, 100x Protease inhibitor</td>
			<td>ThermoFisher</td>
			<td>78429</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Any chemical supplier</td>
			<td></td>
			<td>Laboratory made</td>
		</tr>
		<tr>
			<td>LUNA-FX7 Automated Cell counter</td>
			<td>Logos Biosystems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Any chemical supplier</td>
			<td></td>
			<td>Laboratory made</td>
		</tr>
		<tr>
			<td>N&#176;10 guarded sterile disposable scalpels</td>
			<td>Swann-Morton</td>
			<td>6601</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Sigma Aldrich</td>
			<td>W4502-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Pair of sterile student surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>91401-12</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Any chemical supplier</td>
			<td></td>
			<td>Laboratory made</td>
		</tr>
		<tr>
			<td>RNase Inhibitor 40 U &#181;l-1</td>
			<td>Ambion</td>
			<td>AM2684</td>
			<td></td>
		</tr>
		<tr>
			<td>RNasin 40 U &#181;l-1</td>
			<td>Promega</td>
			<td>N211A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Petri dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>59378-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris buffer, pH 8.0</td>
			<td>Any chemical supplier</td>
			<td></td>
			<td>Laboratory made</td>
		</tr>
		<tr>
			<td>Triton X-100 10% (v/v)</td>
			<td>Sigma Aldrich</td>
			<td>T8787-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Kerloch, T., et al. <a href="https://app.jove.com/v/64369">Fluorescence-Activated Nuclei Negative Sorting of Neurons Combined with Single Nuclei RNA Sequencing to Study the Hippocampal Neurogenic Niche.</a> J. Vis. Exp. (2022).
The video demonstrates a method for isolating intact nuclei from glial and stem cells in the mouse hippocampal dentate gyrus (DG). The DG tissue is mechanically dissociated in a cold homogenization buffer to release the nuclei. Tissue debris and other released organelles are discarded, and the nuclei are stained with a DNA stain and a fluorophore-conjugated antibody specific for the neuronal nuclear antigen (NeuN). Fluorescence-activated nuclei sorting is then employed to isolate the NeuN-negative nuclei of non-neuronal cells.

<img alt="Figure 1" src="/files/ftp_upload/22605/22605_Figure1.jpg" /> 
Figure 1: Preparation of a single nuclei suspension from the dissected DG of adult mice for snRNA-seq of non-neuronal populations. Flow diagram describing the main steps of the protocol that include dissection of mouse DG, preparation of a suspension of single nuclei, NeuN immunostaining, and negative NeuN-FANS-sorting before proceeding with snRNA-seq.
<p class="jove_content" fo:keep-together...

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines
1. Dissection of the DG (Timing: 15 ...

Take dentate gyrus from a mouse brain in a cold homogenization buffer.
Mechanically dissociate to release neurons, glia, and stem cells.
The buffer&#39;s mild detergent permeabilizes the cells, releasing nuclei and other organelles.
Filter the homogenate to remove tissue debris. Then, centrifuge, remove the organelles, and resuspend in the homogenization buffer to lyse any intact cells.
Centrifuge again, remove the supernatant containing released organelles, and resuspend the nuclei in a wash buffer.
Filter to remove any debris, centrifuge, and remove the supernatant.
Introduce a fluorescent DNA stain to label all nuclei and a fluorophore-conjugated antibody that binds to the neuronal nuclear antigen, or NeuN, labeling the neuron nuclei.
Using fluorescence-activated nuclei sorting, identify aggregates with higher DNA stain fluorescence intensity and higher scattering properties, separating the single nuclei.
Analyze the single nuclei, separating the NeuN-positive neuronal nuclei and isolating the NeuN-negative nuclei belonging to the glia and stem cells.

27 ビュー. 海馬歯状回における非神経細胞から核を分離する技術

ビデオ: 海馬歯状回における非神経細胞から核を分離する技術 - 実験

固形腫瘍標本におけるマルチオームシーケンシングのための核の単離

Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data, represents a breakthrough in our ability to discern tumor cell heterogeneity-a primary focus of translational cancer research at this time. However, the quality of sequencing data acquired using this advanced modality is highly dependent on the quality of the input material. 
Digestion conditions need to be optimized to maximize cell yield without sacrificing quality. This is particularly challenging in the context of solid tumors with dense desmoplastic matrices that must be gently broken down for cell release. Freshly isolated cells from solid tumor tissue are more fragile than those isolated from cell lines. Additionally, as the cell types isolated are heterogeneous, conditions should be selected to support the total cell population. 
Finally, nuclear isolation conditions must be optimized based on these qualities in terms of lysis times and reagent types/ratios. In this article, we describe our experience with nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor specimens. We provide recommendations for tissue digestion, storage of single-cell suspensions (if desired), and nuclear isolation and assessment.

著者スポットライト:困難な腫瘍微小環境におけるマルチオームシーケンシングのための核単離の強化

The protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multiome sequencing using the 10x Genomics platform, including recommendations for tissue dissociation conditions, cryopreservation of single-cell suspensions, and assessment of isolated nuclei.

マルチオームシーケンシングのための新鮮および凍結固形腫瘍標本からの最適化された核分離(英語)

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) ...

JoVE Journal

がん研究

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.

蛍光活性化核選別を用いた新鮮凍結皮質からの成人ヒトアストロサイト集団の単離

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular ...

神経科学

Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method was performed for the subsequent paired interrogation of the cell-specific ovarian transcriptome and epigenome using a novel transgenic NuTRAP mouse model. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes, the NuTRAP expression system was targeted to stromal cells using a Cyp17a1-Cre driver. The induction of the NuTRAP construct was specific to ovarian stromal fibroblasts, and sufficient DNA and RNA for sequencing studies were obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with an available Cre line.

In this protocol, the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method were optimized for the paired interrogation of the cell-specific ovarian transcriptome and epigenome using the NuTRAP mouse model crossed to a Cyp17a1-Cre mouse line.

A Technique to Isolate Nuclei from Non-neuronal Cells in the Hippocampal Dentate Gyrus

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A Technique to Isolate Nuclei from Non-neuronal Cells in the Hippocampal Dentate Gyrus