我々は、このプロトコルで使用されているGFP標識、卵白アルブミン特異的T細胞ラインをご提供するためDr。アレキサンダーフリューゲル（マルティンス、ドイツ）を感謝。

1。卵白アルブミン特異的Tリンパ球の刺激安静時の卵白アルブミン特異的T細胞が抗原提示細胞（APC）のような照射ルイスラットの胸腺細胞（30 Gy）をの存在下でオボアルブミンで刺激され、刺激のミディアムで（DMEM +ペン/連鎖球菌/ L -グラット+ NEAA + RPMIのビタミンで準備+ 2ME +ラット血清+オボアルブミン、最終濃度は、材料のリストに記載。） ラット血清の準備<ol><li>我々は胸腺ドナーとして犠牲にラットから血清を取る。 </li><li>ラットではハロタンの吸入により深く麻酔し、できるだけ多くの血液を10 mLシリンジとラットはすぐに死を確保するために断頭されてから23 G 1&quot;針を、使用して心臓の灌流（ラットあたり5〜10 ml）で描画されます。 </li><li>血液は37℃で10分間凝固されている° Cは、氷上で10分間続きます。 </li><li>血栓を除去し、残留液がスピンしている。 </li><li>血清は-20℃で滅菌濾過し、保存され、収集され</li></ol>抗原提示細胞の調製<ol><li>滅菌器を使用して首を切らラットの胸腺を取り出し、氷上でPBSを含むペニシリンとストレプトマイシン（PBS - PS）、に入れてください。 </li><li>血栓や他の​​混入組織からそれをきれいにし、PBS - 10 mlを含む10cmペトリ皿に配置されたセルストレーナー（フィッシャー＃08-771-2）に小さな断片で、それをカット、組織培養フードに胸腺を取るPS。 </li><li>各ピースは滅菌1 mlのシリンジプランジャの背面を使用してセルストレイナーを通して押されている。氷上で50 mlチューブに、単一の細胞懸濁液を収集する。 </li><li> PBS - PSの20-40よりmlのセルストレーナーを洗浄します。 1250グラムで、懸濁液を遠心し、氷上でPBS - PSのペレットは、5ミリリットル/胸腺を、懸濁します。 </li><li>ガンマ照射装置で、この懸濁液（30 Gyを）照射し、直後に50ミリリットルのPBS - PSで2回洗浄する。 注：最高の胸腺のドナーが古い、若い、成人、ラット、5-7週間です。 10週間までのラットを使用されるが、細胞の数は時間とともに減少することができます。一部の人は男性がより胸腺ドナーであるということを言っているものの、性別は重要ではありません。すべて私たちの動物が同じ部屋に収容されているので、我々は常に女性を使用し、我々は同じ部屋で男性と女性を望んでいない。 </li></ol>刺激<ol><li>一度、Tリンパ球を洗浄し、それらを数える。 </li><li>抗原提示細胞を数える。 </li><li>ミックス3 × 10 6卵白アルブミン特異的刺激の培地10mlを含む10cmペトリ皿で150 × 10 6のAPCとT細胞。 </li><li> 37℃で48時間インキュベート℃に</li></ol> 2。卵白アルブミン特異的Tリンパ球の養子移入<ol><li>我々は受信者として使用するラットは7-10週齢の雌です。 </li><li> Tリンパ球をカウント、彼らは大規模で円形にする必要があります。抗原提示細胞が死んでいるだろう。 </li><li> Tリンパ球を遠心し、PBSで10 × 10 6細胞/ mlで再懸濁します。 </li><li> 3ミリリットル注射器と23 G 1&quot;針を使用して1 mlの腹腔内に注入する。 </li></ol> 3。耳の中の抗原と課題<ol><li>二日養子移入した後、他の耳に1つの耳と生理食塩水（または無関係な抗原）で1 mg / mlの卵白アルブミン溶液（=関連抗原）の20μlを注入する。あなたが炎症反応を高める凝集体を失う可能性があるため、抗原溶液をフィルタリングしない。 注：ラットが動きを避けるために麻酔する必要があります。あなたが右利きの場合、左手の親指と人差し指、耳の内側にいる人差し指とあなたが直面しているラットの間に耳をホールド。 </li><li> 27G ½&quot;針を使用して耳の耳介のソリューションを注入する。厳密に同じ場所ですべてのラットを注入してみてください。 </li></ol> 4。 DTHの測定<ol><li>耳は赤と炎症の兆候としてうねり回転を開始します。バネ付きマイクロメートル（例モデルミツトヨからPK - 0505用）をそれぞれの耳の厚さを測定します。 </li><li>各時間点で各耳5-6の測定を行い、平均を計算する。 </li><li>最大の腫れは、24および72時間の間に発生しなければなりません。 </li></ol>

<ol>
	<li>Beeton, C., Barbaria, J., Devaux, J., Benoliel, A. -. M., Gola, M., Sabatier, J. -. M., Bernard, D., Crest, M., Beraud, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+blocking+of+voltage-gated+K++channels+treats+experimental+autoimmune+encephalomyelitis+and+inhibits+T-cell+activation.">Selective blocking of voltage-gated K+ channels treats experimental autoimmune encephalomyelitis and inhibits T-cell activation.</a> J. Immunol. , 166-936 (2001).</li><li>Beeton, C., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+monitoring+of+active+delayed+type+hypersensitivity+(DTH)in+rats.">Induction and monitoring of active delayed type hypersensitivity (DTH)in rats.</a> J Vis Exp. (5), (2007).</li></ol>

抗原投与はまた、背中の皮膚（2）で、例えば、他のサイトで行うことができます。我々は、しかし、それは挑戦見つける。としてこのビデオに示すように耳の耳介を、使用して、DTH反応の最も正確な測定が可能になります。

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		<th>Catalogue Number</th>
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<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco/Invitrogen</td>
<td>12430-062</td>
<td>500ml</td>
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<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S8636</td>
<td>1 mM final, in Stimulation medium</td>
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<tr>
<td>Penicillin &#x2013; Streptomycin &#x2013; L-Glutamine</td>
<td>&nbsp;</td>
<td>Cambrex/BioWhittaker</td>
<td>17-718R</td>
<td>100 u/ml - 100 &micro;g/ml - 4 mM respectively, final, in Stimulation Medium </td>
</tr>
<tr>
<td>Non essential amino acids</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R7131</td>
<td>NEAA, 1% final, in Stimulation Medium</td>
</tr>
<tr>
<td>RPMI vitamins</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R7256</td>
<td>1% final, in Stimulation medium</td>
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<td>beta-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M6250</td>
<td>2ME, 50 &micro;M final, in Stimulation Medium</td>
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<td>Syngeneic rat serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1% final. See explanation in Protocol.</td>
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<td>Ovalbumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5503</td>
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induction and monitoring of adoptive delayed-type hypersensitivity in rats

遅延型過敏症（DTH）は、免疫系がプライミングされているに対して、抗原の注射の部位に侵入CCR7 -エフェクターメモリーTリンパ球によって媒介炎症反応です。炎症反応は、抗原投与の部位の発赤や腫れが特徴です。それは、免疫抑制剤のin vivo有効性の判断に便利なモデルです。皮膚DTHは、抗原特異的Tリンパ球の養子移入によってまたは抗原、および特定の皮膚領域での炎症反応を誘導する抗原とその後の皮課題と能動免疫のいずれかによって誘導することができる。 DTH反応は、例えばオボアルブミン、ツベルクリン、破傷風トキソイド、またはキーホールリンペットヘモシアニンのために、種々の抗原に誘導することができる。そのような反応もMBP、多発性硬化症（1）のための動物モデルによる実験的自己免疫性脳脊髄炎誘発のラットに塩基性タンパク質（MBP）をミエリンに、例えば、自己抗原に対して誘導することができる。ここでは、ルイスラットにおける養子DTH反応を誘導する方法を示します。我々はまずin vitroでの卵白アルブミン特異的T細胞を刺激し、ナイーブなラットにこれらの活性化細胞の腹腔内に注入します。他の耳のウィルは、生理食塩水を受信しながら細胞は2日間、生体内で平衡させた後、我々は、片方の耳の耳介のオボアルブミンをラットに挑戦する。炎症反応は、3から72時間後に表示されますし、耳の厚さは、DTH重症度の指標として測定されます。

Watch this Scientific Journal Video about Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats at JoVE.com

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

遅延型過敏症（DTH）はCCR7 -エフェクターメモリーT（TEM）リンパ球によって媒介炎症反応です。ここでは、抗原特異的なTEM細胞を活性化Lewisラットで養子DTHを誘導し、炎症反応を監視する方法を示します。

ラットにおける養子遅延型過敏症の誘導とモニタリング

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

induction-monitoring-adoptive-delayed-type-hypersensitivity

Research

JoVE Journal

Biology

11.8K ビュー.  University of California, Irvine (UCI). 遅延型過敏症（DTH）はCCR7 -エフェクターメモリーT（TEM）リンパ球によって媒介炎症反応です。ここでは、抗原特異的なTEM細胞を活性化Lewisラットで養子DTHを誘導し、炎症反応を監視する方法を示します。

ビデオ: Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin (KLH).
Here we demonstrate how to induce an active DTH reaction in Lewis rats. We will first prepare a water-in-oil emulsion of KLH, our antigen of interest, in complete Freund's adjuvant and inject this emulsion subcutaneously to rats. This will prime the immune system to develop memory T cells directed to KLH. Seven days later we will challenge the rats intradermally on the back with KLH on one side and with ovalbumin, an irrelevant antigen, on the other side. The inflammatory reaction will be visible 16-72 hours later and the red and swollen area will be measured as an indication of DTH severity.

Induction and Monitoring of Active Delayed Type Hypersensitivity DTH in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes. Here we demonstrate how to induce active DTH in Lewis rats and monitor the inflammatory response.

ラットにおけるアクティブ遅延型過敏（DTH）の誘導とモニタリング

生物学

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7- effector / memory T lymphocytes. Here, we demonstrate a method for inducing and recording the progress of a DTH reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the CCR7- effector / memory T cell response.
An adoptive DTH is induced by the intraperitoneal injection of GFP-labeled Ova-specific CCR7- effector / memory T cell line (Beeton, C J. Visualized Experiments, Issue 8). Cells are then allowed to equilibrate in the rat for 48 hours before challenge by injecting one ear with saline (control ear) and the other with a 1:1 mix of Ova and Ova conjugated to Texas-Red (Ova-TR) to allow visualization of resident antigen-presenting cells.
We describe a method of tissue preparation useful for imaging the motility of cells within the deep dermal layer during an immune response, in conjunction with visualization of collagen fibers by second harmonic generation. Ear tissue is cut into 5 x 5 mm squares (slightly larger is better) and mounted onto plastic cover slips using Vetbond&trade;, which are then secured using silicone grease in an imaging chamber and superfused by oxygen-bubbled tissue culture medium at 37&deg;C.

Here we demonstrate a method for inducing and recording the progress of a delayed type-hypersensitivity (DTH) reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the effector / memory T cell response.

養子DTHの誘導後の耳のイメージングエフェクターメモリーT細胞

Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7- effector / memory T lymphocytes. Here, we demonstrate a ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

鶏胚における神経誘導のためのアッセイ

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

ラットにおける乳腺の形態と乳癌リスクの変化

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

細胞培養で誘導し、低酸素のテスト

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

甲殻類との長期致死毒性試験アルテミアfranciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

ヒト細胞におけるタンパク質発現の誘導のために修飾されたmRNAのインビトロ合成における

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.