この作品は、国立衛生研究所、助成番号NIH / NCRR P41 - RR 016292（ACERT用）によってサポートされていました。

1。材料本研究で使用したすべての溶媒は試薬グレードであった。 N - tert -ブチル-α- phenylnitrone（PBN）と標準的スピンラベルが2,2,6,6 - テトラメチル-1 - piperinyoxyl（TEMPO）を含むスピントラップは、Sigma社から入手し、供給として使用された。植物酸化防止剤は、スワンソン社米国から商業的に入手した。 2。タバコの煙やフリーラジカルの分析の準備<ol><li>リコピンは、アセトンに溶解しながら、フィルタに酸化防止剤を導入するために、抗酸化物質のピクノジェノールとブドウ種子抽出物は、まず、95％エタノールに溶解した。溶媒量は、抗酸化物質の溶解度に応じて異なっていた。 </li><li> 0.4酸化防止剤の量はmg /フィルターを使用した。彼らはその後、活性炭、10mgので被覆した。この目的のために活性炭を真空下で濾過し、乾燥嫌気的条件下で抗酸化溶液との〜12時間、攪拌した。 </l私&gt; <li>酸化防止剤は、従来のアセテートフィルター（CAフィルタ）に導入した。この目的のために、フィルタは2つの部分に切断した。抗酸化物質は、フィルタの2つの間に挿入し、フィルタを形成するためにテープの一部でラップされたコーティングされた植物 - 抗酸化物質フィルターサンドイッチ（図1A）。この組み合わせのフィルタは、その後、タバコを含むタバコのロッドに装着された。制御フィルタは、無酸化防止剤を除く抗酸化フィルタが追加されたのと同じ方法で行われました。 </li><li>喫煙のシミュレーションに先立って、研究のタバコが解凍され、2日間の最低飽和NaBrのソリューションを使用して一定の湿度環境（20℃、相対湿度60％）に格納されていました。 </li><li>ルーチン分析のための喫煙シミュレーションは、水アスピレーターまたはGAST DOA - P104 - BNの真空ポンプ/エアーコンプレッサー（ベントンハーバー、ミシガン）接続で構成され、図1に示すように、シングルポート喫煙装置を用いて周囲温度で実施したスピントラッピングへ一端が開いた状態でT字路を介してアセンブリ。パフは、パフの間に開いたままに開放端を塞ぐことによって行った。ガス流量は、ポンプと通気孔の間に配置されたバルブを調整することによって、〜2.2 SCFH = 17.5 ml / sの時に流量計とセットにより確認した。 </li><li>研究のタバコは、2秒の持続時間を35 mLのパフのボリュームの条件で燻製された1と同様に、60秒ごとに繰り返す。図に示すように、気相フリーラジカルの定量的な推定のための喫煙シミュレーションは、スピントラッピングシステムと連携して実施した。 1。 </li><li>十強烈なパフ（35ミリリットル/パフ）は各タバコのために採取した。気相フリーラジカルは、ケンブリッジフィルターパッドを介してMCSを渡すことによって回収し、スピントラップ溶液（ベンゼン、2.0 mLの0.05 M PBN）に導入した。 </li><li>最後のパフの後、バブルトラップのソリューションは、同じベンゼンとの最初の体積（2.0 ml）に再調整した。アリコート〜25に移した長さ0 mmの一端に封止さ3mmのIDのガラス管。 </li><li>トラッピングソリューションは、凍結 - ポンプ - 解凍の手順を使用して脱酸素した。それは、液体窒素で凍結され、真空を適用した。それはトラップされた気泡が脱出できるように、アルゴン雰囲気下で融解され、そして再び凍結。このサイクルを3回繰り返した後、チューブは真空下で炎シールであり、さらにESR測定に使用。ベンゼンでも可溶である酸素は、有機ラジカルのESR線を広げるため、この手順が必要です。脱酸素化が劇的に信号対雑音比を向上させます。 </li><li> X -バンドESRスペクトルは、標準的条件下で9.34 GHzの周波数でブルカーEMX分光計で記録した。ほとんどの実験で使用した分光器の設定は次のとおりだった：センターフィールド3312.5G、スキャン幅80G、変調振幅0.5G、時定数82マイクロ秒、スキャン時間40秒。 </li><li>スピントラップ付加体は、いくつかのケースではREQの実験条件、で比較的安定しています通常、〜20分を要した25の蓄積を、uired。しかし、12時間後にベンゼン溶液中でのESR信号の強度は〜5倍に減少した。 </li><li>トラッピング付加体の濃度を定量化するために、その最初の一微分ESRスペクトルが統合されました。結果として得られる吸収スペクトルは、煙に含まれるすす/タール製品に起因する可能性が最も高いブロードシングレットの背景を示しています。 このバックグラウンドを差し引いた後にトラップ付加体の分離されたトリプレットが複数の時間（図2）統合されました。 </li></ol> 3。代表的な結果タバコ生産煙を（気相）の燃焼におけるフリーラジカルのほとんどは、瞬時と不安定である。これらの基を観察するために、スピントラップ技術が採用されている。それはより安定であり、ESR（図1）によって検出することができるスピン付加物にそれらを変換することによって気相フリーラジカルを捕捉。 0.05本研究では、スピントラップソリューションM PBNは、酸素と炭素中心15を分離することが困難な基の混合物である煙の気相フリーラジカルを、収集するために使用されていました。私たちのケースでは、しかしながら、観測された超微細分裂定数N = 13.7GとH = 1.95Gは、主要な製品であることを示唆し、アルコキシルフリーラジカルの付加体（RO.）2をトラップするための対応する値に非常によく似ています。我々は、煙の流れの水分によるものであったその弱いESR信号と（図2）私たちの初期の測定で観測された低再現性を示した。この問題を解決するために、我々はケンブリッジフィルターとスピントラップのソリューションとの間の液体窒素（LN2）トラップを追加しました。 LN 2トラップでは、急速凍結によるMCSの流れから水を除去し、ガラス管の内壁に、それを捕捉することも可能です。これは非常にESR信号を改善し、再現性の高い結果（図3）を可能にした。 トラップされたフリーラジカルの量は、使用して決定したリファレンスサンプル。酸化防止剤なしのコントロールのサンプルについては、TEMPOの濃度が既知のための二重積分スペクトルとの重積分スペクトルの比較により推定されたベンゼンの典型的な付加体の濃度は1.24μM（図2）であった。喫煙中にそれぞれのシガレットを通過する空気の量は約350 mlとするので、これは〜7.1X10 -9 MのMCSの気相におけるフリーラジカル濃度の推定値を与え、気相から捕捉されたラジカルの合計数〜1.5 × 15の全体のタバコの。ガスおよび粒子相の両方を含む全体のタバコの煙のフリーラジカルの総量の推定値は、、〜10 16フリーラジカルは9だった。 メインストリームのタバコの煙のガス相フリーラジカルの植物の酸化防止剤の清掃効果の異なるレベルが観察された。彼らの掃気率を図に発表されました。 4。低いながら、リコピンとブドウ種子抽出物は最も高い率を示したER率は、ピクノジェノール（図4）のために観察された。 <img alt="図1" src="/files/ftp_upload/3406/3406fig1.jpg" /> 図1。スピントラップを使用して、たばこの主流煙（MCS）で気相フリーラジカルを収集するための改良された喫煙シミュレーション設計のダイアグラム。 MCSは、ケンブリッジフィルター（イエローフィルター）、CAフィルターを通して水アスピレーターによって描画され、H 2 Oを除去するために液体窒素のトラップを通過させた気相の製品が最終的にトラップをスピンに行き、スピントラップ溶液を通してバブリング。植物の抗酸化物質がMCSにフリーラジカルを捕捉するためにタバコに接続されている（丸で拡大）従来のアセテートフィルターの2枚の間に配置されました。 <img alt="図2" src="/files/ftp_upload/3406/3406fig2.jpg" /> 図2ベンゼンのスピントラッピング付加物濃度の定量的な見積もりは、最初のインテグラから幅広いバックグラウンドシグナルを差し引くことが必要ですlのスペクトル。スピントラッピング付加のための超微細分裂のパラメータは、H = 1.95 G、N = 13.7Gです。 <img alt="図3" src="/files/ftp_upload/3406/3406fig3.jpg" /> 図3。LN2トラップを介して渡すことは、実質的MCSのスピントラップによって得られたESR信号の品質を向上させます。 <img alt="図4" src="/files/ftp_upload/3406/3406fig4.jpg" /> 図4。 MCSにおけるフリーラジカルの濃度で自然な酸化防止剤の効果。相対的シグナル強度は、次のとおりです。コントロール - 100％、ピクノジェノール - 55％、ブドウ種子エキス - 12％、リコピン - 10％。

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タバコの煙の異なるフリーラジカルスカベンジャーの効果の信頼できる推​​定値は、フリーラジカルの定量的検出のための再現可能なテクニックを必要とします。以前に1、それは非極性溶媒に高濃度のスピントラップ溶液の少量がタバコの煙からフリーラジカルをトラップで最も効果的であることが示されている。タバコの煙は、常に有機化合物の燃焼とトラッピング溶媒中で終わってしまうタバコの残留水分から水蒸気が含まれています。 PBNスピントラップ溶液中の水のこの混合物は、実質的スピントラップ付加体の寿命とそのESR信号の強度を減少させる。液体窒素で冷却したU字管を介して簡単に通過するのMCSによってこの水分を削除すると、劇的に気相ラジカルのある部分も凍結面上に捕捉されていても、我々の実験でESRスペクトルの質を改善。 tを使用する彼の手法は、我々は、タバコのフィルターに導入された天然化合物を別のラジカル捕捉の相対効率を比較した。我々はすぐにタバコのフィルターに組み込む後のリコピンとブドウ種子エキスは、気相のMSCからのフリーラジカルの90％まで捕捉できることがわかった。このような高い清掃能力は、ヘモグロビンとshikonine 8,14のような最も効率的な報告フリーラジカルのプロテクタの間でこれらの安価な容易に入手できる天然化合物をランク付けする。まだ、我々の実験に学んだ自然な酸化防止剤をロードしたたばこフィルターは、室温で貯蔵の週後に清掃能力の顕著な部分を失った。この問題を解決することは、商業的なたばこフィルターにリコピンとブドウ種子抽出物の将来のアプリケーションを刺激することができる。

a protocol for detecting and scavenging gas-phase free radicals in mainstream cigarette smoke

喫煙は、ヒトの癌に関連付けられています。それは、肺がんによる死亡のほとんどは喫煙5,6,7,12によって引き起こされることが報告されている。タバコの煙の粒子相のタバコのタールおよび関連製品は、発がん性と変異原性関連疾患の主要な原因ですが、タバコの煙には、9,10の発ガン性物質の重要なグループとみなされているフリーラジカルを大量に含まれています。フリーラジカルは、有害なタンパク質の構造、脂質およびDNA配列により細胞成分を攻撃し、癌の様々なタイプを開発のリスクを増加させる。吸入ラジカルが肺3のタバコの煙の健康への悪影響の多くに寄与する付加体を生成する。研究は、喫煙によって誘発される損傷のリスクを減少させるためにタバコの煙のフリーラジカルを減らすために行われている。それは、ヘモグロビンとヘム含有化合物が部分的に、反応性窒素酸化物を捕捉できることが報告されている酸化剤とタバコの煙4の発がん性nitrosocompounds。 &quot;バイオフィルター&quot;は、ヘモグロビンで構成され、活性炭はフリーラジカルを捕捉すると、タバコの煙14からのフリーラジカルの90％まで除去するために使用されていました。しかし、費用対効果がないため、それが正常に実用化されていません。別の研究では、シコニン、漢方薬8の構成要素の良好な掃気効率を示した。本研究では、我々はスピントラップを使用してたばこの主流煙（MCS）におけるガス相フリーラジカルのスカベンジ効果のたばこの煙のガス相フリーラジカルを捕捉するためのフィルタシガレットに一般的な天然の抗酸化エキスを導入するためのプロトコルと測定を報告する電子スピン共鳴（ESR）分光法1,2,14。我々は、リコピンとたばこフィルターで、将来のアプリケーションを指すようにブドウ種子抽出物の高い清掃能力を示した。これらのプロの重要な利点pectiveスカベンジャーは、それらがそれぞれトマトやワイン産業の副産物11,13から大量に得ることができることです。

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A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

スピントラッピングESR分光法は、植物抗酸化物質リコピン、タバコの煙のガス相フリーラジカルを捕捉に関するピクノジェノールとブドウ種子抽出物の効果を研究するために使用されていました。

たばこの主流煙のフリーラジカルを検出し、気相を清掃するためのプロトコル

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

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19.2K ビュー.  CDCF-AOX Lab. スピントラッピングESR分光法は、植物抗酸化物質リコピン、タバコの煙のガス相フリーラジカルを捕捉に関するピクノジェノールとブドウ種子抽出物の効果を研究するために使用されていました。

ビデオ: A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

タンパク質、細胞閉じ込めのための二次元加工基板を作成する

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

生物工学

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

マイクロピラーとマイクロ流体デバイス内のバイオフィルムストリーマ形成のためのプロトコル

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

ハイドロゲルナノ粒子プラズマの収穫や少量のタンパク質を検出するための尿

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

全体タバコ煙への器官型3D気管支及び鼻腔組織培養モデルの反復暴露の影響評価

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are encapsulated within the aerogels. However, few reports on encapsulating proteins within sol&#8211;gels that are processed to form aerogels exist. A procedure for encapsulating cytochrome c (cyt. c) in silica (SiO2) sol-gels that are supercritically processed to form bioaerogels with gas-phase activity for nitric oxide (NO) is presented. Cyt. c is added to a mixed silica sol under controlled protein concentration and buffer strength conditions. The sol mixture is then gelled and the liquid filling the gel pores is replaced through a series of solvent exchanges with liquid carbon dioxide. The carbon dioxide is brought to its critical point and vented off to form dry aerogels with cyt. c encapsulated inside. These bioaerogels are characterized with UV-visible spectroscopy and circular dichroism spectroscopy and can be used to detect the presence of gas-phase nitric oxide. The success of this procedure depends on regulating the cyt. c concentration and the buffer concentration and does not require other components such as metal nanoparticles. It may be possible to encapsulate other proteins using a similar approach making this procedure important for potential future bioanalytical device development.

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

This procedure describes how to encapsulate cytochrome c (cyt. c) in silica (SiO2) sol-gels, process these gels to form bioaerogels, and use these bioaerogels to rapidly recognize nitric oxide (NO) through a gas-phase reaction. This type of protocol may aid in the future development of biosensors or other bioanalytical devices.

シトクロムをカプセル化<em&gt; C</em&gt;金属ナノ粒子なしのシリカエアロジェルナノアーキテクチャで気相生物活性を保持しながら、

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

マイクロ コント ローラーは従来のタバコの煙、電子たばこエアロゾル液体抽出物の世代のデバイスを運営

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

剛体の埋め込み固定し、ステンド グラス、全体、マイクロ コンピューター断層撮影によってセクション-無料の 3 D の組織学のためのミリ波スケール標本

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

植物活性酸素種体内はアニオン性セリウム酸化物ナノ粒子の触媒の清掃

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

個々のリポソーム間の組成不均一性を検出するための定量的蛍光顕微鏡ベースの単一リポソームアッセイ

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to&#160;glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly&#160;to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to &#62;10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.

The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron.

オーストラリアシンクロトロンにおける逐次結晶学測定用リピニコ注入プロトコル

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...