The authors would like to acknowledge Lynn Opdenaker at the University of Delaware Center for Translational Research at the Helen F. Graham Cancer Center for technical assistance, as well as Nancy Martin from the Duke University Cancer Institute Flow Cytometry Shared Resource, and Dr. Susan H. Smith for guidance in methods and data collection.

1. Preparation for Tissue Collection (15 &#8211; 30 min)
<ol>
	<li>Fire polish three sets of glass Pasteur pipettes in decreasing diameters and number them &#8220;1&#8221; = approximately 1 mm diameter, &#8220;2&#8221; = approximately 0.75 mm diameter, and &#8220;3&#8221; = approximately 0.50 mm diameter.</li>
	<li>Resuspend lyophilized Enzyme A with all of Buffer A according to the protocol provided with the Neural Dissociation Kit (see Table of Materials). Do not vortex. Aliquot the solution into 50 &#181;l&#160;aliquots and store at -20 &#186;C for later use.</li>
	<li>Make 1 L of myelin removal buffer and vacuum filter for sterilization [myelin removal buffer = 1,000 ml of 0.1 M phosphate-buffered saline (PBS), 2 ml of 500 mM Ethylenediaminetetraacetic acid (EDTA), and 10 g of Bovine Serum Albumin (BSA)].</li>
	<li>Cut nylon mesh sheet into small 1&#160; x 1 inch (2.5 x 2.5 cm) squares that will be used for filtering cellular debris and autoclave in a small beaker covered with aluminum foil.</li>
	<li>Prepare 1 L of wash buffer [wash buffer = 1,000 ml of 0.1 M PBS, 2 ml of 500 mM EDTA, and 10 g BSA].</li>
</ol>
2. Tissue Collection
<ol>
	<li>Turn on water bath to 37 &#186;C.</li>
	<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&#8217;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 keep on ice for tissue dissection.</li>
</ol>
3. Tissue Dissection (45 min &#8211; 1 hr)
<ol>
	<li>Inject the animal with appropriate volume of euthanasia solution (commercial barbiturate solution, e.g., Euthasol), according to Institutional Animal Care and Use Committee (IACUC) guidelines, and perfuse out all blood with cold 0.9% saline solution.
	<ol>
		<li>Administer Rats with an overdose of euthanasia solution.</li>
		<li>Monitor anesthesia via breathing and via toe-pinch. When the animal is sufficiently anesthetized (no response to the toe-pinch and deep, slow breathing), the rat can be perfused.</li>
		<li>To perfuse the rat, cut the chest cavity open with a pair of heavy-duty scissors. Cut the diaphragm to allow access up to the heart. Cut the ribs along both sides up to towards the top of the chest, to allow full access to the heart. At that point, the rib cage can be held off to the side using a pair of hemostats.</li>
		<li>Perform cardiac perfusion by inserting an 18 G needle (for adult rats) attached to an electric pump into the left ventricle of the heart. The pump is drawing up cold 0.9% saline solution.</li>
		<li>Then cut open the right atrium with a small pair of scissors to allow for the blood and liquid perfusate to drain from the body.</li>
		<li>Perfuse rats with approximately 10 ml (for adult rats) of 0.9% saline to remove all peripheral immune cells and factors.</li>
	</ol>
	</li>
	<li>Dissect the desired part of the brain region using sterile razor blades and appropriate dissection, then place the tissue pieces into the cold HBSS without Ca2+ and Mg2+. 
	NOTE: The protocol requires HBSS without Ca2+ and Mg2+ in the early steps of the protocol as these ions may interfere with the enzymes required for neural dissociation.</li>
	<li>After all tissue samples have been collected, return to the clean bench or sterile culture hood for tissue processing.</li>
</ol>
4. 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 4.1 and 4.2 for all sample until all samples have been transferred into a 2ml 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 2.2 and 2.3, 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 &#8220;1&#8221;, 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 &#8220;2&#8221;, triturating up and down 30 times. Avoid generating bubbles.</li>
	<li>Dissociate each tissue sample with Pasteur pipette &#8220;3&#8221;, triturating up and down 30 times. Avoid 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>
5. Myelin Depletion (45 min)
<ol>
	<li>Thoroughly re-suspend the pellet of cells with 400 &#181;l of myelin removal buffer.</li>
	<li>Add specified amount of the myelin removal beads and pipette up and down to thoroughly mix. 
	NOTE: The volume of myelin removal beads that is 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. e.g.,&#160;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, 3 times (3 ml total). Collect all flow-through in a waste container such as the bottom of an empty tip box.</li>
	<li>Position 5 ml polystyrene round bottom tube directly underneath each column in preparation of sample collection.</li>
	<li>When cells have finished centrifugation, aspirate supernatant and thoroughly re-suspend in 500 &#181;l of myelin removal buffer. Immediately apply 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>
6. 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 &#8220;single stain&#8221; controls include: APC-CD11b only, GLT1 only, FITC-Thy1 only, PE secondary antibody only, and the &#8220;blank&#8221; 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, vortex the tubes again, cover the tubes with aluminum foil, and incubate the tubes 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 clumping of the cells and thus clogging of the sorter. Samples can also be filtered using a sterile 80 micron filter just prior to sorting in order to avoid clogging the sorter.
	
	</li>
	<li>Take cells to the sorter on ice. Collect the cell populations in 2 ml nuclease-free centrifuge tubes with 0.5 ml of sterile PBS in each tube.</li>
	<li>After the sort, spin the cells down at 300 x g for 2 min at 4 &#186;C. Aspirate off most of the PBS and flash freeze the cells in the -80 &#186;C freezer until further processing for either RNA extraction (see 1 for further protocol details), DNA extraction (see 2 for further protocol details) or cell lysis for protein analysis.</li>
</ol>

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The authors declare that they have no competing financial interests.

Similar to many other tissues and systems, the brain is comprised of a heterogeneous cell population that functions together to impact behavior. The analysis of gene expression, protein expression, or epigenetic modifications from individual cells within that heterogeneous population has the potential to reveal information about the function of the system as a whole, to identify cellular processes regulating both normal behavior and disease processes, and to provide cell-type-specific targets for potential therapies. It ...

以下に記載されたプロトコルの目的は、細胞型特異的遺伝子発現、タンパク質発現、あるいはエピジェネティックマーカーのその後の分析のために、神経組織の不均質な集団から個々の細胞型を単離することです。脳は、異なる前駆細胞に由来する多くの細胞型で構成され、それは、細胞型特異的な特性及び機能を有します。これらの違いにもかかわらず、これらの異なる神経細胞型は、測定または従来の方法を使用して、細胞型特異的に解釈することは困難で、これらの複数のユビキタスタンパク質の分析を行う類似の受容体および細胞内シグナル伝達分子を発現することができます。神経科学者は、脳内のこれらの異なる細胞型の機能および活性化を識別する必要があり、これは最終的には神経学的および神経精神疾患のためのより具体的な薬剤および治療標的を同定するための最初のステップになるように、彼らは個別に動作に影響を与えることができる方法。 Tにもかかわらず、神経科学研究の彼の包括的な目的は、遺伝子またはタンパク質発現、シグナル伝達分子の活性化、またはDNAにエピジェネティックマーカーの修飾を分析するための脳からの個々の神経細胞型を単離することは困難であっできます。細胞型特異的マーカーの追加の染色と組み合わせた場合、これは適切にタンパク質を染色することができる特異的な抗体に依存しているが、現在使用されている技術のうち、免疫組織化学は、細胞型特異的な様式でタンパク質の発現を同定することができます興味のある、それはin situハイブリダイゼーションでは 、脳内の個々の細胞内メッセンジャーリボ核酸（mRNA）の特定の局在を同定することができる。定量化することは困難であるが、これは、特定の細胞型の同時分析を制限する骨の折れるプロセスであり、のみ関心のある1または多分少数の遺伝子の分析を可能にします。レーザーキャプチャーマイクロダイセクションは、視覚化される細胞の亜集団を単離するためにレーザーを使用して顕微鏡を介して、しかし、このプロセスの時間がかかり、自然と比較的低い収率が著しく、これらの分子の発現はそもそも低い場合は特に、タンパク質またはmRNAレベルのその後の分析を制限することができます。蛍光活性化細胞選別（FACS）は、神経科学の分野では比較的新しい技術は、遺伝子発現1および/ ​​またはエピジェネティックなターゲット2のその後の分析のために脳からの個々の細胞型を単離することです。このプロセスは、細胞型特異的遺伝子発現、タンパク質発現、またはエピジェネティックマーカーのその後の分析のために神経細胞の特定のタイプを分類するために使用することができます。 FACSは、物理的または生化学的特徴3のいずれかに基づいて異なる細胞をカウントし、ソートするために、このような数十年のための癌および免疫学などの医学研究のフィールドの数で使用されています。また、フローサイトメトリー古典特異的な抗体を用いて、セルごとにタンパク質の発現を分析するために使用されているフロー。以下の手順は、分子生物学のエンドポイントの、その後の分析のために個々の細胞型を単離するために、古典的なフローサイトメトリー技術を利用します。フローサイトメーターは、上述した技術への迅速かつ効率的な代替手段になります第二に、数千の細胞を分析することができます。さらに、細胞は、特定のタンパク質（例えば、神経伝達物質受容体）、または2つ以上のタンパク質（特定の細胞型における複数のタンパク質の共局在化）の組み合わせの細胞の発現に基づいて単離することができます。これにより、ユーザは、脳内でそれらの機能を識別するために、それらの分子の特性に基づいて、非常に選択的な神経細胞タイプを単離することができます。 FACSを実施するために、神経細胞は、それらが光ビームと分析のためのレーザを通して単一ファイルを通過するように細胞を搬送し、整列フローセルを通過する単一の細胞懸濁液中に調製されます。コンピュータは、各セルとpからデータを取得します。指定されたパラメータ（サイズ、粒度、および蛍光）の分析のためのヒストグラム上たくさんそれを。これらのパラメータに基づいて、細胞はすぐに記憶し、所望の任意のエンドポイントの、その後の分析のために別々のチューブに選別することができます。以下に記載されているプロトコルは、（分化分子11B、のCD11b抗体のクラスタを使用して）（胸腺細胞抗原1、はThy1抗体を使用して）、ニューロン、アストロサイト（グリアグルタミン酸トランスポーター、GLT1抗体を使用して）、およびミクログリアをソートする3つの抗体を利用しています。以下に説明する、または1つが自分の実験のために隔離したい細胞のタイプに応じて異なる抗体で修飾されたように、このプロトコルを使用することができます。 このプロトコルは、特定の実験のために適切であるかどうかを判断する際に考慮すべきいくつかの注意事項があります。一つの大きな注意点は1つが隔離したい特定の細胞型に関係することができます。このプロトコルでは、使用される3つの抗体は、その細胞外の抗体であります実験者は、このように内部のRNA、DNAおよびタンパク質の完全性を維持し、染色手順の間、無傷の細胞型を維持することを可能にします。それは一つだけで、特定の細胞内で発現されるタンパク質を同定する抗体を使用して、特定の神経細胞型を単離することを望む可能性があります。例えば、1はヒドロキシラーゼをチロシンまたはコリンアセチルトランスフェラーゼに対する抗体を用いたアセチルコリン神経細胞を単離するために抗体を用いてドーパミン作動性ニューロンを分離することができます。これらのタンパク質は、細胞内であり、従って、適切な抗体を用いたその後の染色のために細胞膜の固定および透過処理を必要とします。これは、4,5の前に行われてきたが、このプロセスはかなりこれらの透過性細胞からのRNAまたはDNAの収量を減少させることができます。もう一つの注意点は、すべての抗体は、FACSに適していることであってもよいです。例えば、1は現在、ウェスタンブロットのために非常にうまく機能する抗体、Dを必要と技術を使用してもよいですタンパク質のenaturation。この抗体は、必ずしもタンパク質がこのプロトコルのいずれかの時点で変性されていないので、抗体は、その固有の抗原に結合する方法がないことが指定されたFACSを使用してこれらの細胞型の同定に適していないかもしれません。企業は、抗体が承認されたアプリケーションを特定仕様書を提供しています。抗体は、フローサイトメトリーのために承認されていない場合には、特定の抗体を用いて、このプロトコルを試みてから1を妨げるべきではありません。しかし、人はそれをFACSのために動作することが保証されていないことに注意する必要があります。このプロトコルの第三の注意点は1つが隔離しようとしている細胞の数に関係しています。 FACSは、組織の小さな作品から可能なほとんどの細胞を得るための優れた手法であるが、それは1つが比較的小さな脳の領域から細胞の比較的まばらな集団を単離する場合は、一匹の動物からの収量がすることも可能であり、本質的に低いこと。この場合には、とすることができますその後の分析のために必要な細胞数を得るために同一の治療群の数匹の動物からの脳組織をプールすることが必要。しかしながら、最近の刊行物は、遺伝子発現を分析することが可能であることを示す、より大きなセットソートニューロンから起動ニューロン（5〜6％）の少数の遺伝子発現のその後の分析のためのcDNAの遺伝子標的プレ増幅を使用してい動物6この技術の最後の警告を大量にプールすることなく、神経細胞の小さなサブセットから一つが離れすぎていないセルソーターへのアクセス権を持つべきであるということです。セルソーターは、それを適切に使用するにはかなりの訓練を必要とする複雑な機械です。したがって、これらのマシンは、多くの場合、中核施設で資格のある技術者によって運営されています。また、この手順の目標は、おそらく（、神経組織を解離特異的抗体のためにそれを染色し、直ちに短時間でソータにこれらのサンプルを取ることです半日）。この時間枠は、単離された細胞の生存率および収率を増加させ、後続の処理および分析のための細胞の完全性を維持するのに役立ちます。上述のこれらのパラメータの全てが満たされている場合、FACSは、神経組織からの遺伝子およびタンパク質の細胞型特異的発現を分析するための優れた方法です。 全ての実験は、実験動物の管理と使用に関するデラウェア大学の施設内動物管理使用委員会（IACUC）と健康ガイドの国立研究所に従って行いました。

<table border="0" cellpadding="0" cellspacing="0">
	<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>

using fluorescence activated cell sorting to examine cell-type-specific gene expression in rat brain tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The Importance of Myelin Depletion and Tissue Perfusion
Figure 1 depicts the importance of myelin depletion. Myelin depletion (Steps 5.1 through 5.12 of the protocol above) occurs when the single-cell suspension is incubated in the Myelin Removal Beads, washed, and subsequently passed through the column on the magnetic sorter. The purpose of these steps is to reduce the amount of cellular debris present in each sample. As neurons are dissociated and triturated, it can shear of...

Watch this Scientific Journal Video about Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue at JoVE.com

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

using-fluorescence-activated-cell-sorting-to-examine-cell-type

Research

JoVE Journal

Neuroscience

16.8K ビュー.  University of Delaware. The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

ビデオ: Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髄電気生理学II：細胞外吸引電極の作製

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

神経科学

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, respectively 4,5,6,7 The GABAA channel is a pentameric GABA-gated chloride channel. The channel subunits are grouped into 8 families (&alpha;1-6, &beta;1-3, &gamma;1-3, &delta;, &#949;, &theta;, &pi; and &rho;). Two alphas, two betas and one 3rd subunit form the functional channel 8. By combining studies of sub-type specific GABA-activated single-channel molecules with studies including all populations of GABAA channels in the neuron it becomes possible to understand the basic mechanism of neuronal inhibition and how it is modulated by pharmacological agents.
We use the patch-clamp technique 9,10 to study the functional properties of the GABAA channels in alive neurons in hippocampal brain slices and record the single-channel and whole-cell currents. We further examine how the channels are affected by different GABA concentrations, other drugs and intra and extracellular factors. For detailed theoretical and practical description of the patch-clamp method please see The Single-Channel Recordings edited by B Sakman and E Neher 10.

We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.

ラット脳スライスにおけるGABA活性化シングルチャネルとトニック電流

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

開発神​​経上皮における細胞挙動の高解像度のライブイメージング

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

視床前核の脳深部刺激後のラット海馬における遺伝子発現の変化の解析

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

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.

蛍光活性化細胞ソーティング（FACS）と生鮮・冷凍ラット脳組織からのFos発現ニューロンの遺伝子発現解析

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

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 &#215; 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

生体内で蛍光レポーターを使用してマウス視交叉上核における時計遺伝子発現のモニタリング

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can be cultured in defined media for 16 days in vitro and are amenable to any analyses typically performed on dissociated cultures, including electrophysiological, morphological and survival analyses. The major advantage of these cultures is that specific cell types can be selectively studied in the absence of complex external influences, such as those arising from glial cells or other neuron types. When planning experiments with purified cells, however, it is important to note that neurons strongly depend on glia-conditioned media for their growth and survival. In addition, glutamatergic neurons further depend on glia-secreted factors for the establishment of synaptic transmission. We therefore also describe a method for co-culturing neurons and glial cells in a non-contact arrangement. Using these methods, we have identified major differences between the development of GABAergic and glutamatergic neuronal networks. Thus, studying cultures of purified neurons has great potential for furthering our understanding of how the nervous system develops and functions. Moreover, purified cultures may be useful for investigating the direct action of pharmacological agents, growth factors or for exploring the consequences of genetic manipulations on specific cell types. As more and more transgenic animals become available, labeling additional specific cell types of interest, we expect that the protocols described here will grow in their applicability and potential.

This protocol describes a cell sorting based method for the purification and culture of fluorescent GABAergic or glutamatergic neurons from the neocortex and hippocampus of postnatal mice or rats.

蛍光活性化細胞選別により確立された精製 GABAergic または Glutamatergic ニューロンの初代細胞培養

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

げっ歯類の脳におけるタンパク質発現を測定するために近赤外蛍光と高解像度走査を用いること

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

ラット脳組織の疎水性組織除去法

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

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.