我们感谢为我们提供了GFP标记，卵清蛋白特定的T细胞在本议定书中使用的行博士亚历山大弗留葛尔（Martinsried，德国）。

1。卵清蛋白特异性T淋巴细胞的刺激休眠卵清蛋白特异性T细胞受到刺激照射Lewis大鼠胸腺细胞（30戈瑞）作为抗原提呈细胞（APCs）刺激中等，（准备的DMEM + PEN /链球菌/ L的过剩+ NEAA +的RPMI维生素的存在与卵清蛋白+ 2ME +大鼠血清+卵清蛋白，终浓度指出的材料清单。） 大鼠血清的制备<ol><li>我们从我们牺牲的大鼠胸腺捐助者的血清。 </li><li>大鼠深深氟烷吸入麻醉和尽可能多的血液是由心脏灌注，用10毫升的注射器和23政1针，之后将大鼠立即断头，以确保死亡（每大鼠5-10毫升）绘制。 </li><li>血是允许10分钟的血块在37 ° C冰10分钟。 </li><li>血块被删除，剥离剩余的液体。 </li><li>收集血清，无菌过滤和储存在-20 ° C。 </li></ol>抗原提呈细胞的制备<ol><li>一个断头使用无菌文书大鼠胸腺和地方在用含青霉素和链霉素（PBS - PS），在冰上。 </li><li>以胸腺一个组织文化罩，清除血块和其他污染组织，并进入细胞过滤器放置在一个10厘米的培养皿含有10 ML的菜（费舍尔＃08-771-2），切小块的PBS PS。 </li><li>通过细胞过滤器使用1毫升无菌注射器推杆背面的每一块按下。收集到50毫升管上冰的单细胞悬液。 </li><li> 20-40多毫升的PBS - PS冲洗细胞过滤器。离心机1250克悬挂和重新悬浮在PBS - PS颗粒，5毫升/胸腺，在冰上。 </li><li>辐射这个悬挂在伽马辐照（30戈瑞），应立即用50毫升PBS - PS的两倍。 注：最好的胸腺捐助者是年轻成年大鼠5-7周龄。至10周的大鼠可被使用，但细胞的数量随着时间的推移而降低。 性别并不重要，虽然有些人说，男性更好的胸腺捐助者。 我们总是用女性，因为我们所有的动物都在同一个房间内，我们不希望在同一房间内的男性和女性。 </li></ol>刺激<ol><li>一次洗的T淋巴细胞和计数。 </li><li>抗原提呈细胞计数。 </li><li>混合3 × 10 6 150 × 10 6装甲运兵车在10厘米的培养皿刺激培养基中含有10毫升菜的卵清蛋白，特定的T细胞。 </li><li> 48小时在37 ° C。 </li></ol> 2。卵清蛋白特异性T淋巴细胞过继转移<ol><li>我们作为接受者的老鼠是7-10周的女性。 </li><li> T淋巴细胞计数，他们应该是大而圆。抗原提呈细胞将死亡。 </li><li>离心机的T淋巴细胞，并在10 × 10 6细胞/毫升的PBS重悬。 </li><li>注入1毫升腹腔注射3毫升的注射器和23政1“针。 </li></ol> 3。在耳边具有抗原性的挑战<ol><li>继转移后两天，一只耳朵和生理盐水在另一只耳朵（或不相关的抗原）注入20μL1 mg / ml的卵清蛋白溶液（=相关抗原）。不过滤的抗原的解决方案，您可能会失去增强炎症反应的聚合。 注：大鼠麻醉，以避免动作。如果你是右撇子，保持您的左手拇指和食指，食指，耳内，面对你的鼠之间的耳朵。 </li><li>注入使用27G半“针耳耳廓的解决方案。尝试注入完全在同一个地方所有的老鼠。 </li></ol> 4。测量的DTH <ol><li>耳朵将开始变成红和肿胀炎症的标志。衡量一个弹簧加载的千分尺（例如从三丰模型PK - 0505）每耳的厚度。 </li><li>在每个时间点每个耳朵5-6测量和计算平均值。 </li><li>最大的肿胀发生24小时和72小时之间。 </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>

抗原的挑战，也可以在其他网站，例如背部的皮肤（2）。不过，我们发现，具有挑战性的。在此视频中显示，使用耳的耳廓，允许DTH反应的最精确的测量。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco/Invitrogen</td>
<td>12430-062</td>
<td>500ml</td>
</tr>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S8636</td>
<td>1 mM final, in Stimulation medium</td>
</tr>
<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>
</tr>
<tr>
<td>beta-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M6250</td>
<td>2ME, 50 &micro;M final, in Stimulation Medium</td>
</tr>
<tr>
<td>Syngeneic rat serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1% final. See explanation in Protocol.</td>
</tr>
<tr>
<td>Ovalbumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5503</td>
<td>10 &micro;g/ml final, in Stimulation Medium.</td>
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induction and monitoring of adoptive delayed-type hypersensitivity in rats

迟发型超敏反应（DTH）是一个CCR7的效应记忆T淋巴细胞浸润针对其免疫系统已催芽的抗原注射部位介导的炎症反应。炎症反应的特点是由抗原挑战的部位的皮肤发红和肿胀。这是一个方便的模型，以确定在体内的免疫抑制剂的疗效。可诱发皮肤直接到户，抗原特异性T淋巴细胞过继转移或与抗原的主动免疫，并随后在给定面积的皮肤，诱发炎症反应的抗原皮内挑战。 DTH反应，可诱发各种抗原，如卵清蛋白，结核菌素，破伤风类毒素，或锁孔戚血蓝蛋白。这种反应也可以对自身抗原诱导，例如，髓鞘碱性蛋白（MBP）在大鼠实验性自身免疫性脑脊髓炎，多发性硬化症（1）动物模型的MBP诱导。在这里，我们将演示如何在Lewis大鼠诱发领养的DTH反应。我们将首先在体外刺激卵清蛋白特异性T细胞，注入这些天真大鼠腹腔激活细胞。允许细胞以平衡体内2天之后，我们将挑战同在一耳耳廓的卵清蛋白的老鼠，而另一只耳朵WIL收到生理盐水。炎症反应将是可见的3-72小时后，耳厚度测量的DTH严重性的迹象。

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

迟发型超敏反应（DTH）是CCR7的效应记忆T（TEM）淋巴细胞介导的​​炎症反应。在这里，我们将演示如何激活抗原特异性的透射电镜细胞，诱导Lewis大鼠收养的DTH和监测的炎症反应。

收养大鼠迟发型超敏反应的感应和监测

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 次观看.  University of California, Irvine (UCI). 迟发型超敏反应（DTH）是CCR7的效应记忆T（TEM）淋巴细胞介导的​​炎症反应。在这里，我们将演示如何激活抗原特异性的透射电镜细胞，诱导Lewis大鼠收养的DTH和监测的炎症反应。

视频: 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.

大鼠主动迟发型超敏反应（DTH）的诱导和监控

科研

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.

成像效应在耳朵上的记忆体T细胞诱导后收养的DTH

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.

在鸡胚神经诱导的含量为

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.

变化在大鼠乳腺形态和患乳腺癌的风险

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.

在细胞培养中缺氧诱导和测试

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.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

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.

致命的甲壳类动物长期毒性试验卤虫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.

诱导上皮和分析间质转化

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.

在体外合成在人体细胞修饰蛋白表达的mRNA的感应

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.