我々は、ATPアッセイにおける試薬の量を節約するという考えのためJuliann Jaumotteを認める。私たちは、これらの研究のための財政支援を提供するためのメアリー·カルーソ、デブウィルソン、ジャッキーファラーの見事な行政支援のため、製薬マイラン学校に深く感謝しています。おかげでまたHunkele恐怖の病財団とパーキンソン病と運動障害初代神経研究の彼らの財政支援のための基金によるものである。

著者はいずれも開示する競合はありません。

我々は、すべての3つの生存能力アッセイにおける信号強度が線形でプレーティング密度と相関することを見出した。ただし、すべてのアッセイは、メッキ密度が2倍または1.5倍の変化にも同様に区別されます。 N2a細胞の場合は、赤外線のアッセイは、特に低メッキ密度で、ATPアッセイよりも感度が低い。赤外線アッセイは、ATPよりも感度が低いですが、彼らは、N-アセチルシステインの高度?...

生物科学の中で最も一般的な生存率アッセイは、細胞数が含まれます。これは、2013年4月29日およ ​​び2013年4月30日に、キーワードのいずれかを「 インビトロ 」や「文化」とPubMedの中で登場し、トップ（最新の）200の出版物の分析によって証明されている。これらの刊行物の中でも、手動細胞数の計数を含む23.5％用いる細胞計数アッセイは、自動化された細胞数は、イメージングソフトウェアを使用してカウントし、トリパンブルー排除。生/死アッセイは、これらの刊行物の1％で使用した。 MTT（3 - （4,5 - ジメチルチアゾール-2 - イル）-2,5 - ジフェニルテトラゾリウムブロミド）を用いて、多くの刊行物の代謝生存についてのアッセイは11％であった。文学のこの調査では、細胞数アッセイと組み合わせてMTTなどのアッセイを使用して多くの刊行物はわずか3.5％であったことを示しています。それ自体ずつ生存率アッセイを使用する傾向にもかかわらず、細胞数との組み合わせで細胞機能を評価することは私の細胞を評価するための最良の選択と思われるntegrity。残りの細胞が機能または彼らはよく、1,2に存在しているにもかかわらず、健康的ではない可能性があるため、それ自体で細胞数が十分ではない。逆に、例えばATPなどの機能措置が増加または細胞の数の並列の変化の非存在下で低下することがある。細胞数から代謝読み出しの脱共役は、ATPおよびMTTアッセイは唯一の生存率アッセイとして使用してはならないことを示唆している。今回の報告では、細胞構造および代謝機能の両方を調査する3生存率アッセイは、それ自体でアッセイは余裕がある任意の1以上の細胞の完全性のより包括的なビューに記載されている。 我々のアッセイのうちの2つは、700および800nm​​のチャネルで蛍光を測定する赤外線撮像装置を必要とする。ノイズは、より高い信号対雑音比3につながる、赤外線波長が低い。我々は4.5ログダイナミックレンジと16のビット深度、translatinを持って使用オデッセイイメージャ赤外線の2 16または65,536色合いにグラム。これは、波長ごとに色の2 8または256階調を与える8ビットのカラー画像に対比することができる。したがって、16ビットのイメージングは​​、より微細な分解能を有する。これは、元の赤外線画像は、多くの場合、プレゼンテーションのための公表された報告では緑色（800 nm）を赤（700 nm）を疑似カラーしていることに留意すべきである。オデッセイのイメージャは、一般的に西部劇4-7ウエスタンブロット法のためにと、セル内の両方に使用されます。セル内のホルムアルデヒド固定した細胞上の西部劇は、対象となる任意のタンパク質に対する一次抗体を使用し、赤外蛍光二次抗体で順番にそれらにラベルを付ける。この技術は、リンエンドポイント6のために特に有用であることが知られている。私たちにはセル西部劇では、細胞骨格タンパク質α-チューブリンまたは800 nmのチャネル内の神経細胞の微小管関連タンパク質2（MAP2）のための固定した細胞を染色する。これらのタンパク質は、高い信号対雑音比を得るために十分豊富である。また、700で私たちのプレートを染色DRAQ5染色でサファイア染色で細胞質の核のためのnmのチャネル。細胞骨格タンパク質とDRAQ5 +サファイアの汚れの両方は、このように細胞構造を反映している。 第三の生存率アッセイは、代謝機能を測定し、呼ばれて「セルタイターグロ。「このルシフェラーゼベースのアッセイにおいて、発光値がATPレベルに比例している。 ATPアッセイは一般に8-12生存細胞を定量するために使用される。細胞当たりのATP出力が毒素処置の関数として変化する可能性があるため、アッセイの名の下に単語「力価」を含むことは幾分誤った名称であり、セル番号8に比例して、したがって必ずしもである。 ATPレベルはまた、13概日リズムによって、細胞分裂および14細胞分化15によって影響され得る。 ATPは代謝の堅牢な尺度であるので、それにもかかわらず、ここに示されているATPアッセイは実施が簡単で便利です。西部劇はそのためだけではいずれか1アッセイよりも細胞の完全性のより包括的な画像を生成するセル内の赤外線を補完するために、このアッセイを使用して。それ自体が数をセルでない場合は、生存率16〜21、。

<table border="0" cellpadding="0" cellspacing="0" width="821">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Cell Titer Glo</td>
			<td style="width:112px;">Promega</td>
			<td style="width:93px;">G7572</td>
			<td style="width:507px;">Buy in 100 ml quantities and aliquot, instead of purchasing the more expensive 10 ml quantity. Reconstituted, unused reagents can be refrozen at -20 &#176;C for at least 21 weeks</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">18% Formalin</td>
			<td style="width:112px;">Thermo-Shandon</td>
			<td style="width:93px;">9990244</td>
			<td style="width:507px;">Buying this fixative avoids the weighing out of formaldehyde powders and boiling of the solution; exposure to vapors is thereby minimized</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sucrose</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">S0389</td>
			<td style="width:507px;">It is not essential to add this to formaldehyde solutions but it improves the appearance of the fixed cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Odyssey Block</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">927-40003</td>
			<td style="width:507px;">This fish serum can be bought in bulk and frozen at -20 &#176;C for long term use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Triton-X 100</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">21568</td>
			<td style="width:507px;">We store a stock solution of 10% Triton-X 100 in sterile water at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Phosphate Monobasic</td>
			<td style="width:112px;">Fisher</td>
			<td style="width:93px;">S468</td>
			<td style="width:507px;">One can also buy PBS tablets or 10x PBS solutions, but they are more expensive</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Phosphate Dibasic</td>
			<td style="width:112px;">Fisher</td>
			<td style="width:93px;">S373</td>
			<td style="width:507px;">See above</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Azide (250x)</td>
			<td style="width:112px;">Ricca Chemical Company</td>
			<td style="width:93px;">7144.8-16</td>
			<td style="width:507px;">Do not buy the powder because sodium azide is very toxic. We store all our used antibodies in 1x sodium azide at 4 &#176;C until they become contaminated with debris</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Mouse anti-&#945;-tubulin</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">T5168</td>
			<td style="width:507px;">This antibody is expensive but can be greatly diluted and is highly specific</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Mouse anti-MAP2</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">M9942</td>
			<td style="width:507px;">This antibody is expensive but is highly specific (a prerequisite for In-Cell Westerns)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">800 nm Goat anti-mouse IgG</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">926-32210</td>
			<td style="width:507px;">Other companies also sell infrared secondary antibodies. Be sure to purchase the highly cross-adsorbed antibodies and note that concentrations of IgGs may vary with the source</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">DRAQ5</td>
			<td style="width:112px;">Biostatus</td>
			<td style="width:93px;">DR50200</td>
			<td style="width:507px;">This compound used to be sold by LI-COR at 1 mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sapphire</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">928-40022</td>
			<td style="width:507px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Luminometer</td>
			<td style="width:129px;">PerkinElmer</td>
			<td style="width:189px;">VICTOR3 1420 multilabel counter</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Odyssey Imager</td>
			<td style="width:129px;">LI-COR</td>
			<td style="width:189px;">9201-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Shaker/Mixer</td>
			<td style="width:129px;">Research Products International</td>
			<td style="width:189px;">248555</td>
			<td></td>
		</tr>
	</tbody>
</table>

viability assays for cells in culture

顕微鏡で手動細胞数は、細胞の生存能力を評価する敏感な手段ですが、時間がかかり、従って高価である。コンピュータ化された生存率アッセイは、設備の面で高価であるが、より速く、より客観的な手動の細胞数よりもすることができます。今回の報告は、3つのそのような生存率アッセイの使用が記載されている。これらのアッセイの二つは、赤外線であり、1つが発光性である。両方の赤外線アッセイは、16ビット·オデッセイイメージャに依存しています。つの赤外線アッセイは、細胞質ゾルのためのサファイア染色と組み合わせる核についてDRAQ5染色を使用し、700 nmのチャネルで可視化される。他の赤外線分析、インセルウェスタン、細胞骨格タンパク質（α-チューブリンまたは微小管結合タンパク質2）に対する抗体を使用し、800 nmのチャネルでそれらにラベルを付けます。第三の生存率アッセイは、ATPのために一般的に使用される発光アッセイであるが、我々はコストを節約するための推奨量の四分の一を使用しています。これらの測定は、すべての線状であり、CEの数と相関LLSメッキが、感度が異なる。 3つ全てのアッセイは、時間のかかる顕微鏡を回避し、それによりサンプリング誤差を低減し、ウェル全体をサンプリングする。最後に、アッセイの全ては、容易に短期間内で実行される実験の大きな数字を可能にする、実験の終了の1日以内に完了することができる。しかしながら、それらはすべて細胞数が治療後の信号強度に比例、時には特にセルラATPのために、満たされていないという仮定に留まるという仮定に依存する。細胞は、治療後に増加又はサイズが減少した場合さらに、これは細胞数に影響を与えることなく、信号強度に影響を与える可能性がある。我々は、手動カウントを含む、すべての生存率アッセイは、いくつか補足説明に苦しむと結論が、コンピュータ化された生存率アッセイは、初期投資の価値があること。一緒にすべての3つのアッセイを使用して、細胞の構造および機能の包括的なビューをもたらす。

これらの実験における律速因子は、ATPアッセイは、持続時間が比較的短いほど、赤外線染色である。赤外線アッセイのために、我々は、8つの96ウェルプレート4枚のプレートの各々の驚異的な二つのバッチ（ 図1参照）により1日以内に染色し、走査することができると予想している。この推定は、固定の20分を想定し、洗浄30分、洗浄の30分、続いて2時間一次抗体インキュベーショ...

Watch this Scientific Journal Video about Viability Assays for Cells in Culture at JoVE.com

Viability Assays for Cells in Culture

治療用化合物は、多くの場合、最初の生存率アッセイとin vitroで調べられます。人間の観察者によってブラインド細胞数は、細胞数の小さな変化に非常に敏感であることができますが、機能を評価しないでください。コンピュータ化された生存率アッセイは、ここで説明するように、客観的に構造および機能の両方を評価することができる。

培養中の細胞のための生存率アッセイ

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Research

JoVE Journal

Biology

46.0K ビュー.  Duquesne University. 治療用化合物は、多くの場合、最初の生存率アッセイとin vitroで調べられます。人間の観察者によってブラインド細胞数は、細胞数の小さな変化に非常に敏感であることができますが、機能を評価しないでください。コンピュータ化された生存率アッセイは、ここで説明するように、客観的に構造および機能の両方を評価することができる。 

ビデオ: Viability Assays for Cells in Culture

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

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

のてんかんの変異体における発作のためのアッセイをNeurocircuitショウジョウバエ</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

使用シュワン-アストロサイト相互作用の解析インビトロアッセイ

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

ラット肝細胞の単離及び初代培養

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

腎細胞内のミトコンドリアの機能と細胞生存率の評価を過剰発現するプロテインキナーゼCアイソザイム

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

のリフォールディング監視タンパク質を結合アッセイ<em&gt;サッカロミセス·セレビシエ（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

仙台ウイルスのヒト体細胞からの効率的な次世代ヒューマン誘導多能性幹細胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

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 ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

ゼブラフィッシュにおける腎毒素マイクロインジェクションは急性腎障害をモデルに

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

細胞におけるミスフォールドタンパク質の分解のためのアッセイ

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...