数字のサンプルの培養を設定するためのイラストを準備し、カリフォルニア大学ロサンゼルス校（UCLA）クリスReddiするためにはIROCデザインでコリ·サンダース氏に感謝します。このプロジェクトの資金は、HHMI（ハワードグラント番号52006944）によって提供されていました。

<ol>
	<li>Barker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> At the Bench: A Laboratory Navigator. , (1998).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosafety in Microbiological And Biomedical Laboratories (BMBL). , (2009).</li><li>Bykowski, T., Stevenson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+Technique.">Aseptic Technique.</a> Current Protocols in Microbiology. Appendix 4, Appendix 4D (2008).</li><li>Cot&#233;, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+Technique+for+Cell+Culture.">Aseptic Technique for Cell Culture.</a> Current Protocols in Cell Biology. Chapter 1, Unit 1.3 (2001).</li><li>Grimes, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+basic+laboratory+manual+for+the+small-scale+production+and+testing+of+I-2+Newcastle+disease+vaccine.">A basic laboratory manual for the small-scale production and testing of I-2 Newcastle disease vaccine.</a> RAP Publication. AC802/E, 12-13 (2002).</li><li>Guzman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pipetting:+A+Practical+Guide.">Pipetting: A Practical Guide.</a> The American Biology Teacher. 63 (2), 128-131 (2001).</li><li>Jordan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> RESEARCH: National Genomics Research Initiative Phage Resource Laboratory Manual. , (2008).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning - A Laboratory Manual. , 978-087969576 (2001).</li><li>Seidman, L. A., Moore, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic Laboratory Methods for Biotechnology: Textbook and Laboratory Reference. , (2000).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PIPETMAN P User's Guide. , (2012).</li></ol>

私は、開示することは何もありません。

無菌実験室での不要な微生物によって汚染されたなってから滅菌溶液と文化を防止するために行わルーチンプロシージャのセットを指します。このような技術は成長した細胞を必要とする実験のために不可欠です。完全に無菌である職場環境を実現することはできませんが、このように、研究室の表面を消毒ブンゼンバーナーを使用して滅菌フィールドを作成して、空気に上限なしの文化やメディアの露出を制限するなど、ボトル、チューブ、ガラスピペットなどの材料を殺菌するなどの手続き、と非滅菌表面と滅菌の楽器との接触を避けることが実験でソリューションや文化を汚染の可能性を減らすことができます。目標は、第二の天性になるため、これらの予防措置のためのものです。実験室での作業中に、これはトレーニングや練習が付属しています。 このような血清ピペットやマイクなどの楽器を使用して滅菌溶液と文化を持つボリュームの転送ropipettorsは、実験室で行わルーチンのテクニックの多くの種類の一つです。異なる実験アプリケーションは別個の、まだ正確で精度の高いボリュームを転送できる機器用に呼び出します。 micropipettorsは、ソリューションの唯一のマイクロリットル量を必要とする分子生物学的な実験に不可欠である一方血清ピペットは、ミリリットルボリュームを含むメディアの準備を必要とする細胞培養を準備する微生物学研究室で使用されています。無菌技術はこ​​れらの機器を用いて実施される場合は、汚染が液体または実験の種類の量にかかわらず、ボリューム転送中に最小化されます。 このプロトコルで説明されていませんが、一般的に汚染を防止するために使用されるもう一つの手段は、層流フード（ 図10）内で動作するようです。この装置は、組織培養用とBLS-2以上に分類される微生物で行った実験のために重要です。層流フードでは、HEPA（高効率が含まれていますワークスペースに浸透してから部屋からフィルタリングされていない空気を防止しながら、フードに流れる空気から空気中の汚染物質を除去する微粒子空気）フィルター。火炎からの熱は、フードの機能に不可欠な空気の流れを中断させるため注目すべきは、ブンゼンバーナーは、層流フード内では使用できません。 それは、実験を実行するときは、無菌の品質をチェックすると便利です。ソリューションと培地は実験操作中に汚染されないことを確認するには、常にネガティブコントロールを用意します 。たとえば、細菌培養の成長のための培養液のチューブを準備する場合は、1つのチューブだけで滅菌メディアを残して接種していません。そのような意図せずにチューブに導入し、不要な細胞の増殖から濁りなどの汚染の兆候が未接種の対照チューブを点検して接種したチューブと一緒に培地をインキュベートします。制御管が汚染されている場合、実験的な管の可能性が高いだけでなく、汚染され、実験が繰り返されなければなりません。これらの予防措置は、すべての実験を行う必要があります。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> LBブロス</td><td>ディフコ</td><td> 244620 </td><td>基準6でまた利用できるレシピ</td></tr><tr><td colspan="4"> TEバッファー： </td></tr><tr><td> EDTA二ナトリウム塩二水和物</td><td>シグマ</td><td> E5134 </td><td></td></tr><tr><td> Trizma-HClを</td><td>シグマ</td><td> T-3253 </td><td></td></tr><tr><td> CiDecon </td><td>ディーコン·ラボラトリーズ社</td><td> 8504 </td><td>消毒剤</td></tr><tr><td>エタノール</td><td>フィッシャー·サイエンティフィック</td><td> A406 </td><td>消毒剤として使用するために、蒸留水で70％（v / v）を準備する</td></tr></tbody></table>

aseptic laboratory techniques: volume transfers with serological pipettes and micropipettors

微生物があふれている - 空気、土壌、人間の体内で同様に実験室でのベンチやコンピュータのキーボードのような無生物表面上に。微生物の普遍性は、実験室における潜在的な汚染物質の大量の供給を作成します。実験的な成功を確保するために、機器や作業表面上の汚染物質の数は最小限に抑える必要があります。微生物学の多くの実験の間で一般的な細菌細胞またはウイルス粒子を含む培養物の測定と移転技術である。 （1）非滅菌表面に接触または無菌のメディアが必要を汚染することなくこれを行うには無菌スペース、（2）正確に設定し、正確に液体の無菌転送のためのインストゥルメントを読み込み、（3）適切に操作する楽器、文化フラスコ、ボトルと準備滅菌フィールド内チューブ。これらの手順を学ぶことは訓練と実践を求めている。最初に、アクションは、低速意図的、および無菌tに対してれることを目標に制御する必要がありますベンチで作業するとき第二の天性になってechnique。ここでは、ブンゼンバーナーで作成された無菌のフィールド内の血清ピペットとmicropipettorsを使用してボリュームを測定するための手順を紹介します。ボリュームは、マイクロリットル（μL）から使用する機器に応じて、ミリリットル（ml）の範囲です。一般的に転送され、液体は、無菌培養液または薬液など細菌培養とファージ株が含まれています。これらの手順を実行することで、学生のことができるようになります。 <ul><li>ブンゼンバーナーの炎によって作成された無菌のフィールド内で動作します。 </li><li>機器の無菌性を損なうことなく、血清学的ピペットを使用しています。 </li><li>正確にピペットで卒業マークに液体によって形成されるメニスカスを合わせて校正されたボリュームを読んで、血清ピペットで液体を吸引除去する。 </li><li>液体の転送中に培養瓶、フラスコ、試験管、それぞれのキャップは無菌に保つ。 </li><li>プラスチック製の対ガラス血清PIPの異なるアプリケーションを識別ettes。 </li><li> micropipettorsの状態の精度には限界が。 </li><li>正確にかつ正確にmicropipettors上のボリュームを設定します。 </li><li>適切に正しいボリュームを吸引し、転送するピペットに1番目と2番目のストップを使用する方法を知っています。 </li></ul>

Watch this Scientific Journal Video about Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors at JoVE.com

Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors

実験室で作業している場合は、汚染の発生源を最小限に抑えることが不可欠です。無菌非無菌の表面との接触を避けながら、文化や試薬の転送を許可する手順を指します。血清ピペットとmicropipettorsは、実験に使用される溶液の無菌性を損なうことなく、正確なボリュームを測定するために使用されます。

無菌室のテクニック：血清ピペットMicropipettorsのボリュームを転送

Microorganisms are everywhere - in the air, soil, and human body as well as on inanimate surfaces like laboratory benches and computer keyboards. The ...

aseptic-laboratory-techniques-volume-transfers-with-serological

Research

JoVE Journal

Biology

161.8K ビュー.  University of California, Los Angeles. 実験室で作業している場合は、汚染の発生源を最小限に抑えることが不可欠です。無菌非無菌の表面との接触を避けながら、文化や試薬の転送を許可する手順を指します。血清ピペットとmicropipettorsは、実験に使用される溶液の無菌性を損なうことなく、正確なボリュームを測定するために使用されます。

ビデオ: Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors

Microbial Communities in Nature and Laboratory - Interview

自然と実験室での微生物群集 - インタビュー

生物学

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

パッチピペットおよびプログラマブルプラーとシャープ電極作成

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

改善されたパッチクランプ記録のための圧力 - 研磨ピペット

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

回路基板と生体膜のモデル化と軸索の電気信号の測定：学生研究室の演習を

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

Aseptic Laboratory Techniques: Plating Methods

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: 
 
&#9679; Perform plating procedures without contaminating media. 
&#9679; Isolate single bacterial colonies by the streak-plating method. 
&#9679; Use pour-plating and spread-plating methods to determine the concentration of bacteria. 
&#9679; Perform soft agar overlays when working with phage. 
&#9679; Transfer bacterial cells from one plate to another using the replica-plating procedure. 
&#9679; Given an experimental task, select the appropriate plating method.

When working with media and reagents used to culture microorganisms, aseptic technique must be practiced to ensure contamination is minimized. A variety of plating methods are routinely used to isolate, propagate, or enumerate bacteria and phage, all of which incorporate procedures that maintain the sterility of experimental materials.

無菌実験技術：めっき法

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies ...

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

3次元組織学ボリュームとマウス乳腺研究への応用再建

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

フローサイトメトリーを用いて細胞外小胞の分析のための技術

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

マウスで圧力ボリュームループを測定します

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

誘導および定量化細胞老化するテクニック

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

Volume Segmentation and Analysis of Biological Materials Using SuRVoS (Super-region Volume Segmentation) Workbench

Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of interest. When considering the analysis of complex biological systems, the segmentation of three-dimensional image data is a time consuming and labor intensive step. With the increased availability of many imaging modalities and with automated data collection schemes, this poses an increased challenge for the modern experimental biologist to move from data to knowledge. This publication describes the use of SuRVoS Workbench, a program designed to address these issues by providing methods to semi-automatically segment complex biological volumetric data. Three datasets of differing magnification and imaging modalities are presented here, each highlighting different strategies of segmenting with SuRVoS. Phase contrast X-ray tomography (microCT) of the fruiting body of a plant is used to demonstrate segmentation using model training, cryo electron tomography (cryoET) of human platelets is used to demonstrate segmentation using super- and megavoxels, and cryo soft X-ray tomography (cryoSXT) of a mammalian cell line is used to demonstrate the label splitting tools. Strategies and parameters for each datatype are also presented. By blending a selection of semi-automatic processes into a single interactive tool, SuRVoS provides several benefits. Overall time to segment volumetric data is reduced by a factor of five when compared to manual segmentation, a mainstay in many image processing fields. This is a significant savings when full manual segmentation can take weeks of effort. Additionally, subjectivity is addressed through the use of computationally identified boundaries, and splitting complex collections of objects by their calculated properties rather than on a case-by-case basis.

Volume Segmentation and Analysis of Biological Materials Using SuRVoS Super-region Volume Segmentation Workbench

Segmentation of three-dimensional data from many imaging techniques is a major bottleneck in analysis of complex biological systems. Here, we describe the use of SuRVoS Workbench to semi-automatically segment volumetric data at various length-scales using example datasets from cryo-electron tomography, cryo soft X-ray tomography, and phase contrast X-ray tomography techniques.

ボリューム分割と SuRVoS (超領域ボリューム分割) ワークベンチを用いた生物学的材料の分析

Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of ...