نشكر ديك فولي وكيمبال لي لدورهم الرئيسي في تصوير وإنتاج أشرطة الفيديو.

<ol>
	<li>Novak, E. J., Liu, A. W., Nepom, G. T., Kwok, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+Class+II+tetramers+permit+detection+and+characterization+of+peptide-specific+human+CD4++T+cells+proliferating+to+influenza+A+antigen.">MHC Class II tetramers permit detection and characterization of peptide-specific human CD4+ T cells proliferating to influenza A antigen.</a> J. Clin. Invest. 104, R63-R67 (1999).</li><li>Novak, E. J., Liu, A. W., Gebe, J. A., Falk, B., Nepom, G. T., Falk, B., Koelle, D. M., Kwok, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetramer-guided+epitope+mapping:+rapid+identification+and+characterization+of+immunodominant+CD4++T+cell+epitopes+from+complex+antigens.">Tetramer-guided epitope mapping: rapid identification and characterization of immunodominant CD4+ T cell epitopes from complex antigens.</a> J. Immunol. 166, 6665-6670 (2001).</li></ol>

فهم دور خلايا CD4 + T في الحصانة من المهم بشكل أساسي. ومع ذلك ، يمكن CD4 + محددة مستضد الخلايا التائية يكون من الصعب اكتشاف وعزل باستخدام الطرق التقليدية. في المقابل ، MHC الدرجة الثانية tetramers سماح التصور المباشر لخلايا CD4 + T مع خصوصية المستضد المطلوب. هذا الفيديو يدل على العز...

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		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Class 2 Laminar flow biosafety cabinet</td>
<td>equipment</td>
<td>Thermo Forma</td>
<td>1200</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>50 mL conical Tube</td>
<td>equipment</td>
<td>VWR</td>
<td>47747-182</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>1X PBS (Ca/Mg free)</td>
<td>Reagent</td>
<td>HyClone</td>
<td>SH30028.02</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Ficoll-paque PLUS</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1440-03</td>
<td>foil wrapped to protect from light</td>
</tr>
<tr>
<td>Pipet Aid XP</td>
<td>equipment</td>
<td>Drummond</td>
<td>4-000-101</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>10 mL pipet</td>
<td>equipment</td>
<td>Corning/Costar</td>
<td>CLS4492</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Aerosolve Canisters</td>
<td>equipment</td>
<td>Beckman</td>
<td>359481</td>
<td>with 50 mL inserts</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>equipment</td>
<td>Beckman</td>
<td>GS-6R</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Transfer pipets </td>
<td>equipment</td>
<td>Samco</td>
<td>202-20S</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Pasteur pipets</td>
<td>equipment</td>
<td>VWR</td>
<td>14672-200</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Hemolytic Buffer</td>
<td>Reagent</td>
<td>N/A</td>
<td>N/A</td>
<td>8.3 g/L NH4Cl, 1.0 g/L NaHCO3, 0.04 g/L disodium EDTA</td>
</tr>
<tr>
<td>Trypan blue</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T6146</td>
<td>0.2% in PBS</td>
</tr>
<tr>
<td>Hemocytometer</td>
<td>equipment</td>
<td>VWR </td>
<td>15170-208</td>
<td>&nbsp;</td>
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<td>15 mL conical tube</td>
<td>equipment</td>
<td>VWR</td>
<td>05-527-90</td>
<td>Or equivalent</td>
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<td>Running Buffer</td>
<td>Reagent</td>
<td>N/A</td>
<td>N/A</td>
<td>PBS + 2mM EDTA + 5g/L BSA</td>
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<td>MACS CD4+ T Cell Isolation Kit II</td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-091-155</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>EasySep&reg; Magnet</td>
<td>equipment</td>
<td>Stem Cell Technologies</td>
<td>18000</td>
<td>		</td>
</tr>
<tr>
<td>5 mL polypropylene tube</td>
<td>equipment</td>
<td>Falcon</td>
<td>352063</td>
<td>		</td>
</tr>
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<td>RPMI 1640</td>
<td>Reagent</td>
<td> Invitrogen</td>
<td>22400-089</td>
<td>with 25 mM HEPES</td>
</tr>
<tr>
<td>Pooled human serum</td>
<td>Reagent</td>
<td>N/A</td>
<td>N/A</td>
<td>drawn from healthy donors, heat inactivated and filtered</td>
</tr>
<tr>
<td>Pen Strep</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>1570-063</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>500mL 0.22 micron bottle top filter</td>
<td>equipment</td>
<td>Nalgene</td>
<td>595-3320</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>48-well plate</td>
<td>equipment</td>
<td>Costar</td>
<td>3548</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>37&deg;C CO2 incubator</td>
<td>equipment</td>
<td>Sanyo Scientific</td>
<td>MCO-18AIC</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>20 mg/mL synthetic peptide</td>
<td>Reagent</td>
<td>N/A</td>
<td>N/A</td>
<td>Custom synthesis from any peptide vendor</td>
</tr>
<tr>
<td>5 mL FACS tube</td>
<td>equipment</td>
<td>Falcon</td>
<td>352008</td>
<td>Polystyrene</td>
</tr>
<tr>
<td>Peptide loaded class II tetramer (as described)</td>
<td>Reagent</td>
<td>N/A</td>
<td>N/A</td>
<td>Prepared by the BRI tetramer core</td>
</tr>
<tr>
<td>Anti-CD3</td>
<td>Reagent</td>
<td>BD pharmingen</td>
<td>347344</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Anti-CD4</td>
<td>Reagent</td>
<td>eBioscience</td>
<td>17-0049-73</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Anti-CD25</td>
<td>Reagent</td>
<td>eBioscience</td>
<td>11-0259-73</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Facs Calibur Flow Cytometer</td>
<td>equipment</td>
<td>BD Biosciences</td>
<td>342975</td>
<td>Or equivalent</td>
</tr>		</tbody>
		</table>
		</div>

visualizing antigen specific cd4+ t cells using mhc class ii tetramers

معقد التوافق النسيجي الكبير (MHC) من الدرجة الثانية tetramers سماح التصور المباشر لمستضد معين CD4 + الخلايا التائية التي التدفق الخلوي. هذا الأسلوب يعتمد على التفاعل بين الببتيد محددة للغاية ومحملة MHC المقابلة مستقبلات الخلايا تي. في حين أن تقارب من جزيء MHC / الببتيد واحدة منخفضة ، عبر ربط مجمعات MHC / الببتيد مع streptavidin يزيد الطمع من التفاعل ، مما يتيح استخدامها في تلوين الكواشف. بسبب الترددات المنخفضة نسبيا من خلايا CD4 + T (~ 1 في 300000 لخصوصية واحدة) هذا الاختبار يستخدم لتضخيم المختبر في خطوة لزيادة عتبة الخمسين من الكشف. وتنقيته من الخلايا الدموية المحيطية وحيدات النوى التي تقوم عليها Ficoll. ثم يتم فصل خلايا CD4 + عن طريق الانتقاء السلبي باستخدام biotinylated كوكتيل الأجسام المضادة والمعادية للالبيوتين المسمى الخرز المغناطيسي. استخدام خلايا ملتصق من الكسر خلايا CD4 كما مستضد تقديم الخلايا ، CD4 + T يتم توسيع الخلايا في وسائل الإعلام عن طريق إضافة الببتيد مستضدي وايل - 2. هي ملطخة الخلايا موسعة مع tetramer الدرجة الثانية المقابلة التي يحتضنها عند 37 مئوية لمدة ساعة واحدة وملون في وقت لاحق باستخدام الأجسام المضادة السطحية مثل CD4 المضادة ، ومكافحة CD3 ، ومكافحة CD25. بعد وضع العلامات ، ويمكن تحليل الخلايا مباشرة من التدفق الخلوي. الخلايا tetramer شكل إيجابي عادة سكانية متميزة بين CD4 + توسيع الخلايا. خلايا Tetramer ايجابية وعادة ما تكون CD25 + CD4 ، وارتفاع في أغلب الأحيان. لأن مستوى تلطيخ tetramer الخلفية يمكن أن تختلف ، ينبغي دائما أن تكون النتائج ايجابية تلطيخ مقارنة تلطيخ الخلايا نفسها مع tetramer غير ذي صلة. اختلافات متعددة من هذا الاختبار الأساسية ممكنة. ويمكن فرز الخلايا Tetramer إيجابية لمزيد من التحليل المظهري ، أو إدراجها في ELISPOT المقايسات انتشار الأسلحة النووية ، أو فحوصات ثانوية أخرى. وقد أثبتت عدة مجموعات كما شارك في تلطيخ tetramers به ​​وإما لمكافحة خلوى أو أضداد FoxP3.

Watch this Scientific Journal Video about Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers at JoVE.com

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

هذا الإجراء يدل على تنقية والتوسع في المختبر من خلايا CD4 + محددة مستضد الخلايا التائية من الدم المحيطي كله والتصور وذلك باستخدام MHC tetramers الطبقة الثانية Tetramers تسمح التصور المباشر للخلايا T مع خصوصية واحدة ومحددة مستضد MHC تقييد الدرجة الثانية.

المستضد CD4 + تصور معين باستخدام خلايا T MHC الدرجة الثانية Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

visualizing-antigen-specific-cd4-t-cells-using-mhc-class-ii-tetramers

Research

JoVE Journal

Biology

21.6K Views.  Benaroya Research Institute. هذا الإجراء يدل على تنقية والتوسع في المختبر من خلايا CD4 + محددة مستضد الخلايا التائية من الدم المحيطي كله والتصور وذلك باستخدام MHC tetramers الطبقة الثانية Tetramers تسمح التصور المباشر للخلايا T مع خصوصية واحدة ومحددة مستضد MHC تقييد الدرجة الثانية.

Video: Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

CD4 + T - اللمفاويات التقاط باستخدام رقاقة ميكروفلويديك المتاح لفيروس نقص المناعة البشرية

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

عزل خلايا CD4 + T من الغدد الليمفاوية باستخدام الماوس Miltenyi MACS تنقية

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

تقنية لتصور في الوقت نفسه للفيروسات محددة CD8 + الخلايا T والخلايا المصابة بالفيروسات في الموضع</em

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

تحليل خلية معينة من<em&gt; ل arabidopsis</em&gt; يترك طريق الإسفار خلية المنشط الفرز

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

تصور البكتيريا في نيماتودا باستخدام المجهر الفلورسنت

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

A تجليد الفحص إنتاجية عالية MHC II لتحليل الكمي لالببتيد الحواتم

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

الكشف عن التعبير الجيني بين الخلايا في الخلايا الحية من الفئران، الإنسان ولحم الخنزير المنشأ عن طريق النانوية المسمى الإسفار

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

تتبع وقياس العمليات التنموية في<em&gt; C. ايليجانس</em&gt; استخدام أدوات المصدر المفتوح

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin&#39;s lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.

عزل خلايا CD4+ خلايا تي و "تحليل لتعميم" مساعد تي الحبيبي (كتفه) "خلية مجموعات فرعية" من 6-لون "الدم الطرفية استخدام التدفق الخلوي"

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the ...

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

تصور وقياس الأضرار التي لحقت بالحمض النووي الخاصة بالموقع القائم على الإندونوكليز

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is ...