著者らは、マサチューセッツ大学医学部でのフローサイトメトリーコアファシリティで博士リチャードKonzとスタッフによるアドバイスや技術的なご支援を賜りますようお願い申し上げます。糖尿病内分泌学研究センター助成DK032520でサポートされているコアのリソースも使用した。この作品は、国立精神衛生研究所（NIMH）、薬物乱用の国立研究所（NIDA）と母子保健と人間開発の国立研究所からの補助金によってサポートされています。

核抽出<ol><li> douncerに5 mlの溶解バッファー1で冷凍死後の脳組織の250mgを入れ、氷の上に置きます。ソートFACS後の約500万人の核を受信するには、我々は通常、サンプルあたり組織の1000mgのを使用してください。 </li><li>組織が融解した後、しばらく氷上で1分間組織をダウンス。 </li><li>ホモジナイズした組織を取り、15 mLの明確な遠心チューブに入れてください。チューブを氷上に置く。 </li><li> 、ピペットでショ糖溶液2の9 mLを取る明確な超遠心チューブの底にピペットを入れ、ショ糖液2の上にホモジナイズした組織液で濃度勾配を作成するためにショ糖液2を離します。 </li><li>超遠心機の中で回転しながら、それらがすべてバランスされることを確認するために超遠心チューブを秤量する。最上層に溶解バッファー1を添加することにより重量に任意の調整を行う。 </li><li>チューブの重量を測定された後、ローターのバケットに入れます。 </li><li> SW28ローター中にすべてのバケツを置き、慎重に超遠心機にローターを配置。 </li><li> 4℃で2.5時間107163.6 RCFでサンプルを超遠心機℃に</li><li>遠心分離が完了するとチューブの下部にある原子核を含むペレットを乱さないように注意しながら、、真空を使用して、濃度勾配で見つかった残骸の層と一緒に、上清を取り除く。 </li><li>各ペレットに1X PBS 500μLを追加し、20分間氷上に放置。これは、後で簡単に機械的ストレスの量は核の経験を少なくなる、ピペッティングによってそれを溶解すること、ペレットが独自に少しを溶解することができます。 </li><li>原子核は、20分間氷上でインキュベートした後、機械的に完全に1XPBS内核を溶解するために上下にピペッティングし。 </li><li>超遠心管から核を含有する溶液を除去し、単一の2 mLのマイクロ遠心チューブに2本の管の内容を組み合​​わせる。再び、氷の上にサンプルを置きます。 </li></ol> FACSのために免疫染色<ol><li>この時点で、一次および二次抗体、およびブロッキングミックスで構成される免疫染色液を作る。各サンプルについて、NeuN抗体、1XPBSに0.5％BSA、10％正常ヤギ血清を含む、およびAlexa Fluor 488の1μLミックスをブロッキングの100μL、1.2μLを含む1XPBS300μLを組み合わせる。コントロールのサンプルについては、一次抗体のNeuNを除外し、代わりに、単により多くの1XPBSを追加。 </li><li>渦暗所で室温にて5分間、免疫染色混合物とインキュベート。 </li><li>染色混合物がインキュベートされている間、サンプルのコントロールを行います。ソートFACSの場合、サンプルは、単独で核を含む、&quot;未染色のコントロール&quot;が必要とされると呼ばれる、番目のサンプル、ネガティブコントロール、一次抗体のNeuNも必要とされることなく、二次抗体のAlexa Fluor ® 488核を含む。 </li><li>ネガティブコントロール用1XPBS 980 ULを含む別の2 mlチューブに染色制御用1XPBS、そして別の20μLを含む2 mlのマイクロチューブに溶液を含む原子核のアリコートを20μL。核含有1XPBSとバックアップを1 mLのサンプルの音量を上げる。すべてのサンプルは氷上に戻って配置されます。 </li><li>免疫染色の混合物がインキュベート終了したので、適切なサンプルに401μLのその内容を追加します。負のサンプルのみを二次抗体のAlexa Fluor ® 488を含む混合物を受け取る一方核のサンプルは、NeuNおよびAlexa Fluor 488の両方を含む混合物を受け取ります。 </li><li> 45分間暗所で回転するように寒い部屋にサンプルを取る。 </li><li>サンプルは45分間、暗所でインキュベートした後、氷の上に置くとFACSの施設にそれらをもたらす。 FACSの施設への輸送中に、サンプルは氷上で、暗所で保管してください。 </li></ol> FACS <ol><li> FACSの施設で、40ええとフィルタを通してサンプルをフィルタリングする。次に、ソートの前にいくつかの予備データを取得するためには、低温に保たれている間、数分間楽器を介してそれらを実行する。 </li><li>この時点で、それは、ソートに必要な適切なゲートを設定する必要があります。いくつかの異なるゲートは、ソートのサンプルに使用されます。最初のゲートは、サンプル内にある別の原子核の大きさのアイデアを与え、実際の原子核からごみのseparatationすることができます。秒のゲートは、私たちが個々に核をソートすることができます。破棄いかなる凝集体はこの時点で破棄されます。最後に、第3のゲートは、私たちの二次抗体で検出された蛍光色素のことと一致する、特定の蛍光波長に設定されています。このゲートを配置することで、並べ替えることができます、したがって、別のNeuN +の、NeuNから神経細胞の核 - 核。 </li><li>すべてのゲートが選択された後、核が1XPBSに並べ替えることができます。 </li><li>一全サンプルが通過した、楽器によってソートされた試料のアリコートを実行することにより、ソートの純度を確認してください。サンプルの蛍光が広がっていない、のではなく、単一の象限内に含まれていることを確認してください。 90％以上の純粋なサンプルを選択することが最善です。 </li></ol> FACSの後<ol><li>サンプルがソートされると、原子核の処理を開始することができます。ソートされたサンプルの10mLを取り、それにショ糖液2の2 mlを加え、1M CaCl 2及び1メートルマグネシウム（エース）2の30 ulの50μlの。念のため、常に核に修正されていないなので、サンプルを氷上で保管してください。 </li><li>ソートされたサンプルの10未満mLのがある場合は、1XPBSを追加することによって、10mLに音量を上げ上げ、またはそれに応じて追加の材料を調整する。 </li><li>サンプルを15分間氷上で数回と場所を反転。 </li><li>サンプルは15分間氷上でインキュベートした後、4℃で15分間1786 RCFで、スイングバケットローターでそれらを遠心℃に </li><li>チューブの下部にある白いペレットが剥がれないように、真空の使用で上清を捨てる。 </li><li>先に進むの実験に必要なあらゆる溶液200μlにペレットを溶解し、上下にピペッティングし。 </li><li>小さなチューブにソリューションを転送し、さらに使用するまで-80℃の冷凍庫に保存してください。 </li></ol>

<ol>
	<li>Siegmund, K. D., Connor, C. M., Campan, M., Long, T. I., Weisenberger, D. J., Biniszkiewicz, D., Jaenisch, R., Laird, P. W., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+in+the+human+cerebral+cortex+is+dynamically+regulated+throughout+the+life+span+and+involves+differentiated+neurons.">DNA methylation in the human cerebral cortex is dynamically regulated throughout the life span and involves differentiated neurons.</a> PLoS ONE. 2, 895-895 (2007).</li><li>Jiang, Y., Matevossian, A., Huang, H. S., Straubhaar, J., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neuronal+chromatin+from+brain+tissue.">Isolation of neuronal chromatin from brain tissue.</a> BMC Neurosci. 9, 42-42 (2008).</li><li>Spalding, K. L., Bhardwaj, R. D., Buchholz, B. A., Druid, H., Frisen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrospective+birth+dating+of+cells+in+humans.">Retrospective birth dating of cells in humans.</a> Cell. 122, 133-143 (2005).</li><li>Acevedo, L. G., Iniguez, A. L., Holster, H. L., Zhang, X., Green, R., Farnham, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+ChIP-chip+analysis+using+10,000+human+cells.">Genome-scale ChIP-chip analysis using 10,000 human cells.</a> Biotechniques. 43, 791-797 (2007).</li></ol>

ここで紹介するプロトコルは、より一般的にはエピジェネティックな神経細胞クロマチンの変化と、上に、神経核の分子表現型上に、通常の開発と老化の間に、または神経学的または精神疾患に焦点を当てた研究のために特に有用であろう。潜在的な老化の過程でニューロンからグリアの配給量の変化や病気に起因するを含む脳組織の細胞の不均質性は、懸念さ1のときの方法が特に有利で ​​ある。例えば、クロマチン免疫沈降の技術と現在のソートプロトコルを組み合わせることにより、我々は最近、神経栄養因子（BDNF）遺伝子のプロモーター、およびその他の神経細胞の遺伝子のための2由来の脳で神経特異的ヒストンメチル化パターンを説明した。同様のメソッドは、遡及誕生デート3の成人の大脳皮質から神経細胞を収集するために以前に使用されていた。ソート技法は、マウス脳の2で採用され、ために核の安定性の凍結後融解した組織を、我々はここで紹介するアプローチは、種の広い範囲に適用可能であることが予想される。れましたため、わずか10,000細胞のクロマチン免疫沈降法をさらに包括的なゲノムカバー率4で実現可能なので、我々は上記で推奨することを出発物質1000mgのよりもはるかに少ない使用することがあります。

試薬： <table><tr><th colspan="3" align="left"> 1。溶解バッファー</th></tr><tr><td> 0.32M </td><td>スクロース</td><td> 5.47グラム</td></tr><tr><td> 5mMの</td><td> CaCl 2の </td><td> 250μlの</td></tr><tr><td> 3mMの</td><td> MG（アセテート）2 </td><td> 150μlの</td></tr><tr><td> 0.1 mMの</td><td> EDTA </td><td> 10μlの</td></tr><tr><td> 10mMの</td><td>トリス- HCl、PH8 </td><td> 500μlの</td></tr><tr><td> 1mMの</td><td> DTT </td><td> 17μlの</td></tr><tr><td> 0.1パーセント</td><td>トリトンX - 100 </td><td> 50μlの</td></tr><tr><td colspan="3" align="center" style="padding:8px 0 16px 0">オートクレーブした水で50mlにボリュームを調整</td></tr></table><table><tr><th colspan="3" align="left"> 2。蔗糖液</th></tr><tr><td> 1.8 M </td><td>スクロース</td><td> 30.78グラム</td></tr><tr><td> 3mMの</td><td> MG（アセテート）2 </td><td> 150μlの</td></tr><tr><td> 1mMの</td><td> DTT </td><td> 17μlの</td></tr><tr><td> 10 mMの</td><td>トリス- HCl、PH8 </td><td> 500μlの</td></tr><tr><td colspan="3" align="center" style="padding:8px 0 16px 0">オートクレーブした水で50mlにボリュームを調整</td></tr></table><table><tr><th colspan="3" align="left"> 3。バッファをダウンス</th></tr><tr><td> 10 mMの</td><td>トリス</td><td> 0.242グラム</td></tr><tr><td> 4mMの</td><td> MgCl 2の </td><td> 0.163グラム</td></tr><tr><td> 1mMの</td><td> CaCl 2の </td><td> 0.03グラム</td></tr><tr><td colspan="3" align="center" style="padding:8px 0 16px 0"> pHを7.5に調整し、オートクレーブした水を加えて200mlにボリューム</td></tr></table>

neuronal nuclei isolation from human postmortem brain tissue

人間の脳のニューロンは、主に出生前の開発中に分裂となるため、完全な生涯を通じて、その核を維持する。しかし、少しは開発し、老化の過程で、または慢性の精神神経疾患における神経細胞のクロマチンと核組織の変化についてはほとんど知られていない。しかし、ほとんどのクロマチンおよびDNAベースのアッセイ（FISH以外の）不足、単一セルの分解能日付を記入すること。神経細胞の典型的に様々な亜集団がグリア細胞と他の非神経細胞の種類と混ざりされているため、この目的のために、脳組織のかなりの細胞の不均質性は、重要な制限をもたらす。一つの可能​​な解決策は、細胞型特異的な文化を育てることですが、神経細胞を含むほとんどの中枢神経系の細胞は、、数週間で、最高の状態で、持続可能なex vivoのであり、したがって潜在的に完全な寿命にわたって動作エピジェネティックな機構のための不完全なモデルを提供するであろう。ここで、我々は凍結（固定しない）人間の死後の脳から核を抽出し、精製するプロトコルを提供しています。方法は、抗NeuN抗体による超遠心やimmunotagging続いて、低張溶解緩衝液中で核の抽出を行います。標識された神経核は、蛍光活性化ソーティングを使用して別々に収集されます。このメソッドは、種の広い範囲内の任意の脳の領域に適用し、サイトと修正特異的抗ヒストン抗体とクロマチン免疫沈降の研究のための、およびDNAメチル化および他のアッセイに適しているはずです。

Watch this Scientific Journal Video about Neuronal Nuclei Isolation from Human Postmortem Brain Tissue at JoVE.com

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

ほとんどのアッセイは、単一セルの分解能が不足しているため脳組織の細胞の不均質性は、クロマチンにおけるエピジェネティックなマーキングの研究のための重大な制約をもたらす。ニューロンは通常、グリア細胞および他の非神経細胞が混在しています。私たちは、人間の脳から神経細胞核を抽出して収集するプロトコルを提供しています。

人間の死後の脳組織から神経細胞核の単離

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

neuronal-nuclei-isolation-from-human-postmortem-brain-tissue

Nanyang Technological University

Research

JoVE Journal

Biology

22.0K ビュー.  University of Massachusetts Medical School. ほとんどのアッセイは、単一セルの分解能が不足しているため脳組織の細胞の不均質性は、クロマチンにおけるエピジェネティックなマーキングの研究のための重大な制約をもたらす。ニューロンは通常、グリア細胞および他の非神経細胞が混在しています。私たちは、人間の脳から神経細胞核を抽出して収集するプロトコルを提供しています。

ビデオ: Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

人間の脳組織のためのクロマチンアッセイ

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

生物学

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

人間の死後の脳組織における単一細胞から、高品質のRNAを取得

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

マウスおよびヒト結腸組織からの一次筋線維芽細胞の分離

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

からのタグ付き核の親和性に基づく分離<em&gt;ショウジョウバエ</em遺伝子発現解析のための&gt;組織

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

ヒト血清から小非コードRNAの分離

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

マウス膵臓からのRNAの単離：リボヌクレアーゼが豊富な組織

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

Y-27632 成人皮膚組織からのヒトメラノサイトの収穫量を豊かにする

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

洗浄剤と酵素を含まない方法を用いた組織全体からの核分離

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.

ヒト脳組織からのタンパク質の多発反応モニタリングを用いた定量的プロテオミクスワークフロー

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

人間の死後の脳組織から神経細胞核の単離

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