N.G. هو المستفيد من الزمالات من الفرنسية "مؤسسة من أجل Recherche Médicale FRM" ومؤسسة فيليب; R.S. تدعمها مؤسسة لوسيل باكارد لصحة الطفل وجائزة ستانفورد NIH/NCRR CTSA رقم UL1 RR025744; 20- وتحظى شهادة البكالوريوس بدعم من زمالة ماكس كادي من مؤسسة ماكس كادي والأكاديمية النمساوية للعلوم وزمالة شرودينغر من الصندوق النمساوي للعلوم: J3399-B21؛ وزمالة شرودينغر من صندوق العلوم النمساوي( FWF): J3399-B21؛ S.J.G. تعترف بدعم المعاهد الوطنية للصحة منح U19 AI104209, NS 080062 ومن برنامج أبحاث الأمراض المرتبطة بالتبغ في جامعة كاليفورنيا; L.L.R. يعترف بدعم من مؤسسة البحوث الوطنية لالتهاب المفاصل (ANRF) والمعاهد الوطنية للصحة منحة K99AI110645.

وليس لدى صاحبي البلاغ ما يكشفان عنه.

ما يقرب من 30 عاما بعد وصفها الأولي38،'الصاري خلية طرق في'النهج لا يزال يقدم معلومات قيمة حول ما يمكن أن تفعله الشركات متعددة الشخصيات أو لا يمكن القيام به في الجسم الحي. كان يعتقد منذ فترة طويلة أن وظائف MCs تقتصر على دورها في الحساسية. البيانات التي تم إنشاؤها باستخدام'الصار...

الخلايا الصارية (MCs) هي خلايا ما بين الهيماتوبوتية التي تنشأ من سلف نخاع العظم متعدد القدرات1-3. بعد خروج نخاع العظم، تهاجر السلف MCs إلى أنسجة مختلفة حيث تتطور إلى MCs ناضجة تحت تأثير عوامل النمو المحلية1-3. تقع MCs المقيمة في الأنسجة بشكل استراتيجي في واجهات البيئة المضيفة ، مثل الجلد والخطوط الجوية والجهاز الهضمي ، حيث تتصرف كخط دفاع أول ضد الإهانات الخارجية3-6. وغالبا ما تصنف الشركات متعددة الشخصيات على أساس خصائصها الظاهرية "الأساسية" ومواقعها التشريحية. في الفئران، تم وصف نوعين من MCs: "النسيج الضام من نوع" MCs (CTMCs) وMCs المخاطية (MMCs)1-3،7،8. غالبا ما تقع CTMCs حول النولس وبالقرب من الألياف العصبية ، وتقيم في تجاويف مصلية ، في حين تحتل MMCs مواقع داخل الظهارة في الأمعاء والغشاء المخاطي التنفسي1-3.
وقد طبقت منهجيات عديدة لدراسة الوظائف البيولوجية لمركبات الكربون المتعددة المركبات9-13. وقد ركزت مجموعات كثيرة على النهج في المختبر باستخدام خطوط الخلية إما (مثل خطوط MC الإنسان HMC114 أو LAD215,16), في المختبر MCs المستمدة (مثل الإنسان الطرفية المشتقة من الدم MCs17, أو نخاع العظام الماوس-MCs المستزرعة المشتقة [BMCMCs]18، MCs المستزرعة المشتقة من جلد الجنين [FSCMCs]19 و MCs المشتقة من الخلايا الصفاقية [PCMCs]20)أو MCs المعزولة من مواقع تشريحية مختلفة. وتستخدم جميع هذه النماذج على نطاق واسع لدراسة التفاصيل الجزيئية لبيولوجيا MC، مثل مسارات الإشارات المشاركة في تنشيط MC. ومع ذلك ، فإن جانبا هاما من بيولوجيا MCs هو أن خصائصها الظاهرية والوظيفية(على سبيل المثال، محتوى بروتياز الحبيبية السيتوبلازمية أو الاستجابة لمحفزات مختلفة) يمكن تحويرها حسب الموقع التشريحي والبيئة الدقيقة2،7. منذ الخليط الدقيق من هذه العوامل التي تواجهها في الجسم الحي قد يكون من الصعب إعادة إنتاج في المختبر، ونحن نفضل استخدام في نهج في الجسم الحي للحصول على رؤى في وظائف MCs9.
توجد عدة سلالات فأرة مع نقص MC الوراثي ، مثل WBB6F1المستخدم على نطاق واسع -كيت W / W-v أو C57BL /6 - كيت W-sh/W-sh الفئران. هذه الفئران تفتقر إلى التعبير و / أو نشاط KIT (CD117)، مستقبلات عامل نمو MC الرئيسي عامل الخلايا الجذعية (SCF)21،22. ونتيجة لذلك ، فإن هذه الفئران لديها نقص عميق MC ولكن لديها أيضا تشوهات إضافية فينوتيبيك تتعلق بطفراتهاc-kit (في WBB6F1-Kit W / W-v mice) أو بآثار الانعكاس الكروموسومي الكبير الذي يؤدي إلى انخفاض تعبيرc-kit (في C57BL/6 -Kit W-sh/W-sh mice)9,10,12,23. في الآونة الأخيرة ، تم الإبلاغ عن عدة سلالات من الفئران مع جعدة- المستقلة نقص MC التأسيسية24-26. وقد استعرضت مؤخرا كل هذه الفئران وبعض أنواع جديدة إضافية من الفئران MC ناقص غير قابل للانتقاص بالتفصيل9,10,13.
هنا، نقوم بوصف طرق توليد MCs المستزرعة المشتقة من نخاع العظم الماوس (BMCMCs)، ونقلها بالتبني إلى الفئران MC ناقصة، وتحليل أعداد وتوزيع MCs المنقولة بالتبني في مواقع تشريحية مختلفة. يمكن استخدام هذه الطريقة المسماة "طرق الخلايا السارية" لتقييم وظائف MCs والمنتجات المشتقة من MC في الجسم الحي. ونناقش مزايا هذه الطريقة وحدودها، في ضوء النهج البديلة التي تم تطويرها في السنوات الأخيرة.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>1% Antibiotic-Antimycotic Solution</td>
			<td>Corning cellgro</td>
			<td>30-004-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>3 ml Syringe</td>
			<td>Falcon</td>
			<td>309656</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm x 10 mm Dish</td>
			<td>Corning cellgro</td>
			<td>430588</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml Polystyrene Round Bottom Tube</td>
			<td>Falcon</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid Glacial</td>
			<td>Fisher Scientific</td>
			<td>A35-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue 8GX</td>
			<td>Rowley Biochemical Danver</td>
			<td>33864-99-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra 6R Centrifuge</td>
			<td>Beckman</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD16/32 (clone 93) Purified</td>
			<td>eBioscience</td>
			<td>14-0161-81</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 1 ml TB Syringe</td>
			<td>BD Syringe</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 22G x1 (0.7 mm x 25 mm) Needles</td>
			<td>BD Precision Glide Needle</td>
			<td>205155</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 25G 5/8 Needles</td>
			<td>BD Syringe</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 30G x1/2 Needles</td>
			<td>BD Precision Glide</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue MAX Jr, 15 ml Polypropylene Conical Tube</td>
			<td>Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C298-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytoseal 60 Mounting Medium</td>
			<td>Richard-Allan Scientific</td>
			<td>8310-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytospin3</td>
			<td>Shandon</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>DakoCytomation pen</td>
			<td>Dako</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco Modified Eagle Medium (DMEM) 1x</td>
			<td>Corning cellgro</td>
			<td>15-013-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E 7023-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum Heat Inactivated</td>
			<td>Sigma Aldrich</td>
			<td>F4135-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Conjugated IgG2b K Rat Isotype Control</td>
			<td>eBioscience</td>
			<td>14-4031-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein Isotiocyanate (FITC) Conjugated Anti-mouse KIT (CD117; clone 2B8)</td>
			<td>eBioscience</td>
			<td>11-1171-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Fisher Scientific</td>
			<td>F79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Giemsa Stain Modified</td>
			<td>Sigma Aldrich</td>
			<td>GS-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Isothesia</td>
			<td>Henry Schein Animal Health</td>
			<td>29405</td>
			<td></td>
		</tr>
		<tr>
			<td>May-Grunwald Stain</td>
			<td>Sigma Aldrich</td>
			<td>MG-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiwell 6 well plates</td>
			<td>Falcon</td>
			<td>35 3046</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus BX60 Microscope</td>
			<td>Olympus</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast Plus Tissue Embedding Medium</td>
			<td>Fisher Brand</td>
			<td>23-021-400</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Conjugated IgG Armenian Hamster Isotype Control</td>
			<td>eBioscience</td>
			<td>12-4888-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered-Saline (PBS) 1x</td>
			<td>Corning cellgro</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Phycoerythrin (PE) Conjugated Anti-mouse FceRIa (clone MAR-1)</td>
			<td>eBioscience</td>
			<td>12-5898-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide Staining Solution</td>
			<td>eBioscience</td>
			<td>00-6990-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-3</td>
			<td>Peprotech</td>
			<td>213-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Safranin-o Certified</td>
			<td>Sigma Aldrich</td>
			<td>S8884</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flasks T25 25 cm2</td>
			<td>Beckton Dickinson</td>
			<td>353109</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flasks T75 75 cm2</td>
			<td>Beckton Dickinson</td>
			<td>353110</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine Blue 1 % Aqueous</td>
			<td>LabChem-Inc</td>
			<td>LC26165-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse SCF</td>
			<td>Peprotech</td>
			<td>250-03</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing the functions of mast cells in vivo using 'mast cell knock-in' mice

الخلايا الصارية (MCs) هي خلايا دموية موجودة في أنسجة مختلفة ، وهي وفيرة بشكل خاص في المواقع المعرضة للبيئة الخارجية ، مثل الجلد والمسالك الهوائية والجهاز الهضمي. اشتهر دورهم الضار في الحساسية التي تعتمد على IgE ، كما ظهرت MCs كلاعبين مهمين في الدفاع المضيف ضد السم والبكتيريا الغازية والطفيليات. يمكن أن يتأثر النمط الظاهري ووظيفة MC بالعوامل المحيطة الدقيقة التي قد تختلف وفقا للموقع التشريحي و / أو استنادا إلى نوع أو مرحلة تطور الاستجابات المناعية. لهذا السبب، ونحن وآخرون قد فضلت في نهج في الجسم الحي على أساليب في المختبر للحصول على نظرة ثاقبة وظائف MC. هنا، نقوم بوصف طرق توليد المركبات متعددة الثقافات المشتقة من نخاع العظم (BMCMCs)، ونقلها بالتبني إلى فئران ناقصة وراثيا MC، وتحليل أعداد وتوزيع MCs المنقولة بالتبني في مواقع تشريحية مختلفة. وقد استخدمت هذه الطريقة ، التي تسمى'الصاري خلية طرق في' النهج ، على نطاق واسع على مدى السنوات ال 30 الماضية لتقييم وظائف الشركات متعددة الأجهزة والمنتجات المشتقة من MC في الجسم الحي. ونناقش مزايا هذه الطريقة وحدودها، في ضوء النهج البديلة التي تم تطويرها في السنوات الأخيرة.

ويرد في الشكل 1نظرة عامة على نهج'طرق الخلايا السارية'ويشمل توليد مركبات بي إم سي إم سي، وعدد الخلايا التي ينبغي أن تكون محفورة في أي p.p. أو أي أي في الفئران MC-deficient (يمكن أن يختلف العدد إذا أشير إليها على أساس التصميم التجريبي) والفاصل الزمني بين الحرمان والتجربة اعتمادا على موق?...

Watch this Scientific Journal Video about Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice at JoVE.com

Analyzing the Functions of Mast Cells In Vivo Using &#039;Mast Cell Knock-in&#039; Mice

نحن نصف طريقة لتوليد الخلايا الصاري المشتقة في المختبر ، وغرغرتها في الفئران التي تعاني من نقص الخلايا الصاري ، وتحليل النمط الظاهري والأرقام وتوزيع الخلايا الصارية المحتقنة في مواقع تشريحية مختلفة. يمكن استخدام هذا البروتوكول لتقييم وظائف الخلايا السارية في الجسم الحي.

تحليل وظائف الخلايا الصاري في فيفو باستخدام 'الصاري خلية طرق في' الفئران

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such ...

analyzing-functions-mast-cells-vivo-using-mast-cell-knock

Research

JoVE Journal

Immunology and Infection

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

10.2K Views.  Stanford University School of Medicine. نحن نصف طريقة لتوليد الخلايا الصاري المشتقة في المختبر ، وغرغرتها في الفئران التي تعاني من نقص الخلايا الصاري ، وتحليل النمط الظاهري والأرقام وتوزيع الخلايا الصارية المحتقنة في مواقع تشريحية مختلفة. يمكن استخدام هذا البروتوكول لتقييم وظائف الخلايا السار?...

Video: Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

بعد الخلوي في مصير<em&gt; E. القولونية</em&gt; بعد الإصابة التي كتبها لامدا بالعاثية

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

تحريض الطعم ضد المضيف المرض و<em&gt; في الجسم الحي</em&gt; مراقبة الخلايا T باستخدام نموذج MHC المطابقة الفئران

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

والفحص صفيحة ميكروسكوبية لتقييم تأثيرات المواد الكيماوية على RBL-2H3 الصاري تحبب الخلايا: آثار التريكلوسان بدون استخدام مذيب عضوي

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

التحقيق ماست الخلية الإفرازية حبيبات. من البناء الحيوي لالإيماس

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

تقييم الشبكية دبقية أكلة وظيفة<em&gt; في فيفو</em&gt; باستخدام الفحص القائمة على الخلوي التدفق

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

في فيفو الفحص للكشف عن وظيفة محددة مستضد تي خلية عن استخدام نموذج التطعيم

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

عزل خلايا ماست المستمدة من الغشاء البريتوني وعلى التوصيف الوظيفي مع Ca2 +-التصوير وفحوصات تحبب

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

وصف الاستجابات ايستب الغلوبولين المناعي تعتمد على الغدة الصعترية، والغدة الصعترية مستقلة في الفئران باستخدام مقايسة الممتز المرتبط بالانزيم

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

جيل خلايا المغلوب الأساسية والموسعة ناغورني كاراباخ البشرية باستخدام ريبونوكليوبروتينس Cas9

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.