本稿で説明した作業は、パトリック·F·テイラー財団からの資金によって可能になりました。

1。接着細胞の染色体収穫<ol><li> 標準プロトコル <ol><li>特定の細胞培養条件に応じて細胞を増殖させる。細胞が対数増殖期（80％コンフルエント）に達したとき、細胞培養フラスコにコルセミドを10μl/ミリリットル加える。 2×10 6セルの最小を推奨します。 </li><li> 45分間、5％CO 2インキュベーター中37℃で細胞をインキュベートする。滅菌ピペットを使用して、15ミリリットルコニカルチューブに細胞からメディアを転送。置いておきます。 </li><li>穏やかフラスコにHBSS緩衝液2 mlを添加することによって細胞を洗浄する。スワールは、バッファしてからピペットを用いて除去する。捨てる。 </li><li>それがフラスコの表面全体を覆​​うことを確実にする、1mlのトリプシンを加える。わずか約2分間トリプシンで細胞を残す。細胞の大部分が分離したら、戻って細胞上にコニカルチューブ内のメディアをピペット。 </li><li>を15mlコニカルチューブに、10mlの一定分量中の細胞懸濁液を移す。10分間200×gで遠心分離する。上清を除去し、ペレットを再懸濁。 </li><li>コニカルチューブ内の残りのペレットに37℃に温めておいてきた0.075MのKClを10ミリリットルを追加します。 KClおよび細胞を混合する程度の速度で渦管。 </li><li> 10分間37℃で細胞をインキュベートする。 25℃で5分間200×gで遠心分離（約0.5ミリリットル残るまで）上清を除去し、ペレットを再懸濁し。 </li><li>ボルテックスしながら細胞に：慎重に新鮮なカルノア固定液（氷酢酸メタノールの3:1比率）の5ミリリットルを追加。その後、10ミリリットルの合計ボルテックスせずに固定液より5ミリリットルを追加します。 </li><li> 5分間200×gで遠心分離する。上清と再懸濁細胞を除去する。各チューブに固定液の5ミリリットルを追加します。 </li><li> 5分間200×gで遠心分離する。上清と再懸濁細胞を除去する。各チューブに固定液の5ミリリットルを追加します。細胞はこれまでに一年間、4℃で保存することができる。 </li></ol></li><li> 議定書の修正：染色体harvestiリンパ芽球様細胞株のngの<ol><li>特定の細胞培養条件に応じて細胞を増殖させる。 （使用して、培養された細胞の量に適したフラスコ）。細胞は（細胞が分割され、約2日後）対数期に達すると、細胞培養フラスコにコルセミドの10μL/ mLを加え。 </li></ol></li><li> 議定書の修正：全血から染色体収穫 フィトヘマグルチニン（PHA）、金時豆に由来したレクチンは、ヒトT細胞16のための強力なマイトジェンである。 72時間の培養にPHAを添加した後、細胞の約45％がS期にある。これは、ピーク分裂活性を示し、染色体研究のために採取するれる最適点である。 B細胞17,18を分析するとき、例えばヤマゴボウ（1-10μg/ ml）をのような他のマイトジェンを使用することができる。 <ol><li>緑のトップヘパリンナトリウム管内における全血を少なくとも1ミリリットルを収集します。採取後3日以内に使用してください。 RT国連で保存血使用する準備がゴマ。 </li><li>アリコートL-グルタミン（20％ウシ胎児血清、1％ペニシリン/ストレプトマイシン、1％ファンギゾン、および1％PHA）を含有する完全RPMI培地10ml中の全血0.25mlの。 5％のCO 2、37℃で培養する。 </li></ol></li><li> 議定書の修正：骨髄から染色体収穫 染色体異常クローンの観察は、白血病の特定のタイプのための診断され得るように、骨髄での細胞遺伝学的解析は、多くの悪性血液疾患に有用である。染色体の研究はまた、急性転化および治療に対する応答の発症などの疾患の進行を評価するために使用される。骨髄細胞が活発に分裂しているので、まだマイトジェン刺激9,19は不要である。急性白血病のために24および48時間培養も設定されています。 <ol><li>緑のトップヘパリンナトリウム管内における骨髄の少なくとも1ミリリットルを収集します。アリコートL-グルタミンを含む完全RPMI培地10ml中の骨髄0.25mlの（20％ウシ胎児血清、1％ペニシリン/ストレプトマイシン、1％ファンギゾン）。 5％のCO 2、37℃で培養する。 </li></ol></li></ol> 2。スライドの準備とソリッド染色 1代表スライドの迅速な評価は、例えば、G-バンディング、魚、CGH、または空のようさらなる手続きに進む前に、収穫の品質に関する情報を提供します。いくつかの研究室では、固体染色するために迅速な収穫と染色体評価のための細胞を好む。代替的には、位相差顕微鏡は、この分析のために使用することができる。湿度が約50％と温度雰囲気（20〜25℃）である場合、スライドを最も良く調製される。 <ol><li> 25℃で5分間、200×gで細胞を遠心分離唯一の0.3〜0.5ミリリットル残るまで上清を取り除きます。 </li><li>優しくペレットを再懸濁した後、ピペット約45°の角度で傾いたとサスペンションを許可されたスライド上では約2の距離から細胞懸濁液の3滴スライド上を流れていき。スライドに新鮮なカルノア固定液の1大粒を追加します。 </li><li>ペーパータオルの上のスライドの裏側を乾燥した後、少なくとも10分間乾燥さ引き出して座る。スライドは完全に乾燥する必要があります。 </li><li>新鮮ギムザ染色液（ガーバッファおよびギムザ染色の3:1の比）を準備します。染色ラックにスライドを置きます。ギムザ染色液中のスライド全体をカバーしています。スライドを5分間染色溶液中に残存してみましょう。 、蒸留水でスライドをすすいで排水し、空気乾燥させ。 </li><li> 4パーマウント滴のスライドにカバースリップを追加します。カバースリップの下に気泡がないことを確認してください。過剰のパーマウントは、紙タオルで除去することができる。 </li><li> 10Xと100X拡大して光学顕微鏡で細胞を分析します。中期細胞が豊富でよく普及している場合、残りのスライドを、他の実験手順を用いることができる。 </li></ol> 3。トリプシンおよびギムザを用いて、G-バンディング（GTG） &lt;のp&gt;トリプシンは、得られたより高い転写活性を有するDNA領域にユークロマチン、ヒストンを変性タンパク質分解酵素である。ギムザ染色後、これらの領域は、光のバンドとして表示されます。ほとんど、あるいは全く転写活性（ヘテロクロマチン）と高度に凝縮クロマチンをトリプシンから保護そのヒストンの大部分を持つことになりますので、ギムザ染色後暗く染色します。それは、最初にG-バンドのスライドに状況を監視し、必要に応じてその後のスライドでトリプシンタイミングを調整することが不可欠である。 <ol><li> 4コプリンジャーに以下の手段を追加します。 ジャーファー＃1X HBSSの1 = 30ミリリットルと10倍のトリプシン（0.5％）の4ミリリットル 1XのHBSSのJAR＃2 = 50ミリリットル 1X HBSSおよびウシ胎児血清の5ミリリットルのジャーファー＃3 = 45ミリリットル 1X HBSSのJAR＃4 = 50ミリリットル</li><li> 5秒のJAR＃1の各スライド、ジャー＃2の迅速なリンスを浸し、少なくとも30秒間ジャー＃3の各スライドのまま、ジャー＃4クイックすすぎ、その後、スライドを乾燥させます。 </li><li> FRESを準備しますHギムザ染色液（ガーバッファおよびギムザ染色の3:1の比）。染色ラックにスライドを置きます。ギムザ染色液中のスライド全体をカバーしています。スライドを5分間染色溶液中に残存してみましょう。 </li><li>これらは染色されたのと同じ順序でスライドを蒸留水ですすぐ。スライドを約10分間乾燥させます。スライドは完全に乾燥する必要があります。 </li><li> 4パーマウント滴のスライドにカバースリップを追加します。カバースリップの下に気泡がないことを確認してください。過剰のパーマウントは、紙タオルで除去することができる。 </li><li> 10Xと100X拡大して光学顕微鏡で細胞を分析します。最初のスライドの結果に基づいてスライドの残りのトリプシンタイミングを調整する。 </li></ol>

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著者らは、開示することは何もありません。

我々は、種々のヒトから得られた細胞型、アカゲザル、ラットおよびマウス11,20-22を含む種々の生物の細胞から染色体を単離するための本手順を利用している。標準的なプロトコルが提供されるが、特定の重要なステップと変数は、細胞のこれらの多様なタイプのために調整される必要があり得る。いくつかの具体的な手順は、染色体の準備と結果の可能な限り最高の品質を確保する...

染色体分析は、染色体不安定性と遺伝性疾患や悪性腫瘍の1,2,8,9につながる再配列を診断するために、世界的に利用される従来の手法である。また、憲法および癌取得した遺伝的異常の診断および研究のための高解像度は、例えば、蛍光in situハイブリダイゼーション （FISH）などの古典的な細胞遺伝学的手順および分子細胞遺伝学的方法論の組み合わせ、比較ゲノムハイブリダイゼーション（CGH）で達成することができる、およびスペクトル核型分析（SKY）14,15。より最近では、これらの技術は、幹細胞研究に関連付けられた染色体不安定性の評価のために利用されている。そのような長期培養胚細胞（ES）および種々の生物の成体幹細胞の異数性のような核型異常は、複数の研究室により報告されている。最近の証拠は、いくつかの細胞系は本質的に、染色体に対してより傾斜していることを裏付けるinstabi関係なく、培養条件のLITY。確立および/またはヒト、マウス、アカゲザル幹細胞株を維持するときに、この理由のため、染色体分析は、品質管理プロセスの一部として推奨されている。多くの報告が文化10月13日中の幹細胞および悪性細胞または様々な生物の染色体の安定性を監視するためのルーチンおよび分子細胞遺伝学の使用の関心の高まりを説明します。これらのプロトコルは、ますます培養細胞の迅速な13染色体非遺伝的評価のために実験室で使用されている。我々は、様々な生物由来の臨床研究のため、細胞の両方に適用することができる種々の細胞型からの染色体を調製するための我々の基本的な手順を提示する。

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">KaryoMAX Colcemid</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:145px;">15212-012</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS Buffer</td>
			<td>Gibco</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% Trypsin EDTA 10x</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Permount</td>
			<td>Fisher Scientific</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buffer Tablets &#34;GURR&#34;</td>
			<td>Gibco</td>
			<td>10580-013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Geimsa Stain</td>
			<td>Ricca Chemical Company</td>
			<td>3250-16</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge 5810</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>Caledon Laboratory Chemicals</td>
			<td>6700130</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glacial Acetic Acid</td>
			<td>Krackeler Scientific Inc.&#160;</td>
			<td>11-9508-05</td>
			<td></td>
		</tr>
	</tbody>
</table>

chromosome preparation from cultured cells

染色体（細胞遺伝学的）分析は、染色体不安定性の検出のために広く使用されている。 G-バンディングや、in situハイブリダイゼーション（FISH）蛍光として分子技術が続く場合には、このアッセイは、1つ以上の染色体が関与するゲノム再配列の部分の損益を伴う収差を個々の細胞を分析するための強力な能力を持っています。ヒトでは、染色体異常は約1 160あたりの出生1,2、すべての流産の60から80パーセントで発生する3,4、死産の10％の2,5、先天性心疾患6、3から6パーセントを持つ個人の13％不妊のケース2の、そして発達遅滞と先天性欠損7と多くの患者において。クローン性染色体異常の観察は、診断および予後的意義8,9の両方を有することが示されているように、悪性腫瘍の細胞遺伝学的分析は、日常的に、研究者や臨床医によって使用される。60、染色体の分離は、遺伝子治療のための非常に貴重であり、ヒト以外の霊長類とげっ歯類10月13日を含む生物の幹細胞研究。 染色体は、血液リンパ球、皮膚線維芽細胞、羊膜細胞、胎盤、骨髄、および腫瘍検体を含む生体組織の細胞から単離することができる。染色体は、それらが最も凝縮された場合に、有糸分裂の中期段階で分析され、したがって、よりはっきりと見える。染色体分離技術の最初のステップは、後続の後期段階に進むから細胞を防ぐためにコルセミドとのインキュベーションによって、紡錘糸の破壊を伴う。次いで、細胞を低張液で処理し、カルノア固定液との膨潤した状態で保存されている。次いで、細胞をスライドガラス上に滴下し、次いで、種々の手順のために利用することができる。 Gバンド法は、特徴的な明暗の帯を作成するためにギムザで染色し、トリプシン処理を必要とする。同じPR染色体を単離するocedureは、例えば、in situハイブリダイゼーション （FISH）、 蛍光 、比較ゲノムハイブリダイゼーション（CGH）、スペクトル核型分析（SKY）14,15のような手順のための細胞の調製のために使用することができる。

高品質の中期スプレッドは、染色体分析のために不可欠です。成功したアッセイは、よく普及し、適切な染色体の形態である染色体が得られます。適切にG-バンド染色体特性明暗バンドパターンが含まれています。 <img alt="図1" fo:content-width="5in" fo:src="/files/ftp_upload/50203/50203fig1highres.jpg" src="/files/ftp_upload/50203/50203fig1.jpg" /> 図1。染色体?...

Watch this Scientific Journal Video about Chromosome Preparation From Cultured Cells at JoVE.com

Chromosome Preparation From Cultured Cells

染色体は、例えば、リンパ球または皮膚線維芽細胞等の生細胞から、ヒトまたはマウスを含む生物から単離することができる。これらの染色体調製物は、さらに、ルーチンG-バンディングなどsituハイブリダイゼーション （FISH）、 蛍光 、比較ゲノムハイブリダイゼーション（CGH）、スペクトル核型分析（SKY）のような分子細胞遺伝学手順のために利用することができる。

培養細胞からの染色体の準備

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

chromosome-preparation-from-cultured-cells

Research

JoVE Journal

Biology

80.7K ビュー.  Louisiana State University Health Science Center. 染色体は、例えば、リンパ球または皮膚線維芽細胞等の生細胞から、ヒトまたはマウスを含む生物から単離することができる。これらの染色体調製物は、さらに、ルーチンG-バンディングなどsituハイブリダイゼーション （FISH）、 蛍光 、比較ゲノムハイブリダイゼーション（CGH）、スペクトル核型分析（SKY）のような分子細胞遺伝学?...

ビデオ: Chromosome Preparation From Cultured Cells

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

カウントと培養細胞の生存率の決定

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

生物学

Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually scattered cells and small cell groups. This technique uses HistoGel (Thermo Scientific) with conventional laboratory equipment.
The use of cell block sections is a valuable ancillary tool for evaluation of non-gynecologic cytology. They enable the cytopathologist to study additional morphologic specimen detail including the architecture of the lesion. Most importantly, they allow for the evaluation of ancillary studies such as immunocytochemistry, in-situ hybridization tests (FISH/CISH) and in-situ polymerase chain reaction (PCR). Traditional cell block preparation techniques have mostly been applied to non-gynecologic cytology specimens, typically for body fluid effusions and fine needle aspiration biopsies.
Liquid based cervicovaginal specimens are relatively less cellular than their non-gynecologic counterparts with many individual scattered cells. Because of this, adequate cellularity within the cell block sections is difficult to achieve. In addition, the histotechnologist sectioning the block cannot visualize the level at which the cells are at the highest concentration. Therefore, it is difficult to monitor the appropriate level at which sections can be selected to be transferred to the glass slides for testing. As a result, the area of the cell block with the cells of interest may be missed, either by cutting past or not cutting deep enough. Current protocol for Shidham's method addresses these issues. Although this protocol is standardized and reported for gynecologic liquid based cytology specimens, it can also be applied to non-gynecologic specimens such as effusion fluids, FNA, brushings, cyst contents etc for improved quality of diagnostic material in cell block sections.

Shidham's method for preparation of cell blocks with AV-marker from cytology specimens containing individually scattered cells and small cell groups.

個別散在セルの優位を持つ細胞診標本からのセルブロックの準備

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually ...

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

抗体標識用ショウジョウバエ多糸染色体のカボチャの準備

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

生細胞におけるストア作動性カルシウムエントリの蛍光ベースの測定：培養癌細胞から骨格筋線維に

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

培養細胞における小胞体サブドメインの可視化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

培養哺乳動物細胞での超並列レポーターアッセイ

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

培養細胞およびマウス組織から単離とミトコンドリアの機能解析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

イメージングローカルCA<sup&gt; 2+</sup&gt;培養哺乳動物細胞内シグナル

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

植物におけるFISHのための適切な高速空気乾燥滴下染色体製造方法

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

マウス精母細胞から減数分裂期染色体の準備をスプレッドします。

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...