K.ウォン、STCウォンは、機能と分子イメージング研究センター、ブリガムアンドウィメンズ病院からの資金でサポートされています。 K.ウォン、STCウォンは、バイオテクノロジーや情報、メソジスト病院研究所、ワイルコーネル医科大学のためのセンターではサポートされています。 KKクォン、I.チェン、JQ RENは、生体イメージングのためのAthinoula A. Martinosセンター、マサチューセッツ総合病院からの資金によってサポートされています。 Lenuta Kloetzer、ブレーデン郭はNeuroenteric研究センター、マサチューセッツ総合病院からの資金でサポートされています。 著者は、博士に感謝します。 Q.曽と光イメージングラボ、ブリガムと技術的なヘルプのための女性の病院のX.徐。

<ol><li>女性のHO - 1 - ルシフェラーゼトランスジェニックマウス（4-6週齢）は到着後、（株）タコ農場から購入することができる、マウスが宿泊を許可するには、最低4日間、動物の収容施設に配置されます。 </li><li>生物発光イメージングのための準備： <ol style="list-style-type:lower-alpha;"><li>各HO - 1マウスを量る。動物の体重は、ルシフェリンの投与量を計算するために必要です。 </li><li> 脱毛：またはバック動物の腹部および/ ​​上の毛リムーバーNairさん®を適用するために綿のスワップを使用してください。 5〜10秒待って。髪をふき取るためにきれいな綿のスワップを使用してください。クリーン残りの髪を拭くために蒸留水に組織片をつけます。 </li><li>バックグラウンドノイズを低減するためIVIS 100ステーションのイメージングプラットフォーム上で非蛍光黒い紙（ストラスモアArtagain ®黒い紙）を置きます。 </li><li>ルシフェリン（Xenogen社から）7.5 mg / mlの（滅菌H 2 Oに溶解）の濃度で溶液を調製します。ルシフェリンの投与量は65.5 mg / kg体重である。このように、20グラムのマウスには、0.175ミリリットルルシフェリン溶液の腹腔内注射を必要とします。これを達成するために、ルシフェリン溶液（0.175ミリリットル）の単一の注入量は、26ゲージの針で注射器にプリロードされ</li></ol></li><li> 瓜沙手順：瓜沙は、生物発光イメージングプロトコルを実行する前に一度だけ適用されます。瓜沙が苦痛ではないので、マウスだけ冷静にイソフルランによる短時間麻酔にする必要があります。瓜沙の手順は、次の手順が含まれます： <ol style="list-style-type:lower-alpha;"><li>瓜沙で瓜沙の手順の間に数回をターゲットと肌の部分を潤滑するために食用油または蒸留水を適用します。 </li><li>繰り返しセラミックスープ用のスプーンやプラスチックのスプーンを使用して、外柔内剛の力でマウスの背中の毛のない領域を削る。 </li><li>バックスキンは、通常2〜3分以内に達成皮下溢血の記号、である、赤になるまで、スクレイピングは続きます。 </li></ol></li><li> HO - 1の発現上昇が数時間にわたって徐々に構築されるように生物発光イメージングは、瓜沙直後または数時間起動されることがあります。生物発光イメージングのための手順は、次のとおりです。 <ol style="list-style-type:lower-alpha;"><li>イソフルラン（1.5％）、医療グレードの空気の混合物で満たされた麻酔導入チャンバー内にマウスを麻酔。 </li><li>マウスに麻酔をしたら、IVIS 100光学イメージングステーション上のイメージング室へマウスを移動してください。仰臥位（上腹部）にマウスを置きます。イメージングチャンバーは、継続的にイソフルランの1.5％を注入される。イメージングプラットフォームは、° C、マウスを暖かく保つために37℃で加熱される。 </li><li> &quot;ミディアムビニング&quot;で画像の取得と30秒に設定された露光時間を設定します。画像を取得するために開始します。手動または自動で、画像の取得を3分ごとに繰り返すようにマシンを設定します。最初のイメージの取得後は、利息（ROI）の領域は、腹部、胸部、および頭部の領域をカバーするように描画されます。このROIは、コピーされ、&quot;リビングイメージ®&quot;、IVIS 100駅に付随するソフトウェアを使用して次の画像に貼り付けられます。 </li><li>信号強度は毎秒の光子で測定されます。そのピーク値に達すると、ピーク時間後に約5〜10分間の画像上でマウスを保つために信号強度の時間をマーク。 </li><li>仰臥位でのイメージング買収が完了すると、マウスを裏返しにし、腹臥位（バックアップ）でそれを置く。今ソフトウェア&quot;リビングイメージ®&quot;の利用で、背中や頭部の領域をカバーするために描かれたROIと5〜10分のためのイメージング動物を続ける。画像の選択は、仰臥位後に腹臥位は任意です。一つは、興味のある主要な器官に応じて、腹臥位で開始するかを選択できます。別のオプションは、画像に別々の実験では仰臥位となりやすい位置です。 </li><li>腹臥位でのイメージング買収が完了したとき、イソフルランをオフにし、回復するために誘導室にイメージング室からマウスを移動します。誘導室は、現在唯一の医療の空気を注入し、マウスは、通常1分未満で目覚めています。 </li><li>後処理のための画像データを保存します。 </li><li>初期イメージング研究の後、追加の画像の取得は、瓜沙後数日間実施される。一つのサンプルスケジュールは、12時間目 、24 番目の時間、36時間目 、48 番目の時間、72 回目の時間と120時間目でのイメージングを繰り返したが、撮影スケジュールの正確なタイミングは柔軟です。長期のフォローアップが必要な場合は、追加のイメージングセッションが追加される可能性があります。髪が戻って成長している場合はイメージを作成する前に、毛の取り外しの手順を繰り返します。 </li></ol></li><li>対照群の場合は、すべての手順を繰り返しますが瓜沙（ステップ3）をスキップします。マウスの同じグループはコントロールと瓜沙、制御の経験の両方に使用されている場合erimentは、好ましくは瓜SHA手順の前または少なくとも一ヶ月後に行われるべきである。 </li></ol>代表的な結果： 瓜沙への応答におけるHO - 1の生体内で上方制御における図1のshowの生物発光画像。図2のグラフは、瓜沙に関連し、同じマウスの全身から光束（光子/秒）で120時間以上の定量的な経時変化を示しています... <img src="/files/ftp_upload/1385/figure1tmb.jpg" alt="図1" border="0" /> 図1の左からそれぞれ18時間、36時間および120時間後瓜沙で瓜沙の前に、同じマウスの正面図（仰臥位）の右側に、代表的な画像、、へ。瓜沙後、1つは消化管、生殖管、肝臓、腎臓（バックビューから、図示せず）、及び他の領域を包含する多臓器の重要な信号強度の増加の進行状況を観察する。してください<a href="https://www.jove.com/files/ftp_upload/1385/figure1.jpg">ここをクリックして</a>図1の拡大版のために。 <img src="/files/ftp_upload/1385/figure2tmb.jpg" alt="図2" border="0" /> 図2図1の同じマウスで瓜沙の後120時間にわたって追跡全身からフラックスの定量的変化（光子/秒）。してください<a href="https://www.jove.com/files/ftp_upload/1385/figure2.jpg">ここをクリックして</a>図2の拡大バージョンを参照すること。

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The authors have nothing to disclose.

HO - 1、ヘムオキシゲナーゼの誘導型、の転写は、ヘム、過酸化水素、紫外線照射、低酸素症、および物理的ストレスを含む多くの要因によってアップレギュレートされる。そのような報告されたHO - 1の生物発光のプロトコルとして全身イメージングでは、複数の臓器で、全身HO - 1発現のクイックスナップショットを提供します。小動物では、生物発光イメージングは​​、高感度のin vivo全?...

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<td>Luciferin</td>
<td>&nbsp;</td>
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<td>IVIS 100</td>
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bioluminescence imaging of heme oxygenase-1 upregulation in the gua sha procedure

瓜沙は、皮下微小血管の血液の溢出やあざの原因となる皮膚を削ることを採用し、伝統的な中国の民間療法です。 HO - 1 -の生物発光の光イメージングのためのプロトコル<em&gt;ルシフェラーゼ</emこの原稿で報告された&gt;トランスジェニックマウスは、迅速に提供します。<em&gt; in vivoで</em瓜沙プロシージャへの応答におけるヘムオキシゲナーゼ-1（HO - 1）遺伝子発現のアップレギュレーションの&gt;アッセイ。 HO - 1は酸化ストレスに対する細胞保護作用を提供するために、古くから知られている。生物発光の出力により評価されたHO - 1のアップレギュレーションは、、瓜沙によって解放循環ヘモグロビンの製品に抗酸化応答を表すと考えられている。点状出血（スプリンター出血）または斑状出血（あざ）皮下毛細血管から血液の血管外漏出の指標が観察されるまで瓜沙は、バックやトランスジェニックマウスの他の標的の身体の部分で潤滑皮膚上に滑らかなスプーンの端の繰り返しストロークによって投与した。瓜沙後は、生物発光イメージングのセッションは、複数の内臓にHO - 1発現のダイナミクスに従うように数日間、毎日実施した。

Watch this Scientific Journal Video about Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure at JoVE.com

Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

瓜沙、中国の伝統的な治療皮膚を削ることは、皮下微小血管の血液の溢出を引き起こします。我々は、HO - 1 -の生物発光イメージングのプロトコルを報告する<em&gt;ルシフェラーゼ</em瓜沙は、複数の器官におけるヘムオキシゲナーゼ1（HO - 1）アップレギュレートすることを実証するために&gt;トランスジェニックマウス。

瓜沙手続におけるヘムオキシゲナーゼ-1アップレギュレーションの生物発光イメージング

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol ...

bioluminescence-imaging-heme-oxygenase-1-upregulation-gua-sha

Research

JoVE Journal

Biology

13.9K ビュー.  Massachusetts General Hospital, Harvard Medical School. 瓜沙、中国の伝統的な治療皮膚を削ることは、皮下微小血管の血液の溢出を引き起こします。我々は、HO - 1 -の生物発光イメージングのプロトコルを報告するルシフェラーゼトランスジェニックマウス。

ビデオ: Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

A Craniotomy Surgery Procedure for Chronic Brain Imaging

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an extremely useful method, because it allows the study of the live intact brain. With appropriate preparations, this technique allows the observation of the same cortical area chronically, from minutes to months. In this video, we show a preparation for chronic in vivo imaging of the brain using two-photon microscopy. This technique was initially pioneered by Dr. Karel Svoboda, who is now a Howard Hughes Medical Institute Investigator at Janelia Farm. Preparations like the one shown here can be used for imaging of neocortical structure (e.g., dendritic and axonal dynamics),  to record neuronal activity using calcium-sensitive dyes, to image cortical blood flow dynamics, or for intrinsic optical imaging studies. Deep imaging of the neocortex is possible with optimal cranial window surgeries. Operating under the most sterile conditions possible to avoid infections, together with using extreme care to do not damage the dura mater during the surgery, will result in successful and long-lasting glass-covered cranial windows.


This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

慢性脳イメージングのための開頭手術の手順

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an ...

生物学

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

in vitroおよびヒト胚性幹細胞のin vivoでの生物発光レポーター遺伝子イメージングの

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

マウス腎移植の手順

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

FDG micro-PET/CTマウスにおける褐色脂肪の機能イメージング

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

ルシフェラーゼ生物発光レポーターを用いた遺伝子発現のモニタリング細胞自律的な概日時計のリズム

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

アポトーシス細胞のクリアランスのイメージング中に生きる<em&gt;ショウジョウバエ</em&gt;胚

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

生物発光共鳴エネルギー転移を用いて生細胞中のタンパク質 - タンパク質相互作用を調査する

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

哺乳動物細胞におけるヘム合成レベルの測定

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

mtHyper7を用いた生酵母細胞における過酸化ミトコンドリアのイメージング

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for&#160;more consistent expression compared to plasmid-borne constructs.
mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

著者スポットライト:ミトコンドリア研究の進歩 - 細胞内解析のためのmtHyper7バイオセンサー 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

生きた酵母細胞におけるミトコンドリア過酸化物のレシオメトリックバイオセンサーmtHyPer7のイメージング

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...