نشكر الدكتور U. عاشري لهدية من NPY-mRFP [كدنا]. نشكر الدكاترة. MJ Kofron، L. ميتلمان، M. Shaharbani، وY. زلبرشتاين للحصول على مساعدة لا تقدر بثمن مع المجهري وصورة ويحلل. كما نشكر الدكتور جوزيف أورلي لقراءة نقدية لهذه المخطوطة. وأيد هذا العمل من خلال منحة من مؤسسة العلوم إسرائيل، التي تأسست من قبل الأكاديمية الوطنية الإسرائيلية للعلوم (1139-1112 إلى RS-E).

<ol>
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C., Kempuraj, D., Tagen, M., Conti, P., Kalogeromitros, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+release+of+mast+cell+mediators+and+the+pathogenesis+of+inflammation.">Differential release of mast cell mediators and the pathogenesis of inflammation.</a> Immunol Rev. 217, 65-78 (2007).</li><li>Caughey, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell+proteases+as+protective+and+inflammatory+mediators.">Mast cell proteases as protective and inflammatory mediators.</a> Adv Exp Med Biol. 716, 212-234 (2011).</li><li>Lundequist, A., Pejler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+implications+of+preformed+mast+cell+mediators.">Biological implications of preformed mast cell mediators.</a> Cell Mol Life Sci. 68 (6), 965-975 (2011).</li><li>Metz, M., Maurer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cells--key+effector+cells+in+immune+responses.">Mast cells--key effector cells in immune responses.</a> Trends Immunol. 28 (5), 234-241 (2007).</li><li>Gordon, J. R., Burd, P. 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The authors have no financial conflicts of interest.

وصفنا استراتيجية مبتكرة تجمع بين الكمي لإم سي إس إيماس وأربعة (س، ص، ض، ر) quantifications البعد عن طريق ثلاثي الأبعاد التصوير ساقطا وقت جان الدراسات في الخلايا الحية باستخدام الجين مراسل إيماس. هذه التقنية تتيح فحص عائلات البروتينات من حيث أثرها على وظيفة MC مثل جان الدراسات...

الخلايا البدينة (MC) هي خلايا المناعة التي اشتهر مشاركتهم في الحساسية والتهابات مثل التهاب المفاصل والربو والتهاب المريء بالحمضيات، التهاب الجلد المزمن وصدمة الحساسية 1،2 فضلا عن الأمراض الأخرى بما في ذلك مرض الشريان التاجي 3،5 و السرطان 3،4. وبالإضافة إلى ذلك، إم سي إس تلعب أدوارا هامة في المناعة الفطرية والتكيفية، سواء في الدفاع المضيف ضد البكتيريا والطفيليات وقمع الاستجابات المناعية، على سبيل المثال حمل المزروع التسامح 5،6. المعلقون تنشأ من النخاع العظمي، وتطوير من CD34 + / CD117 + الخلايا الاصلية المحفزة 7. يتم الافراج نخاع العظام ملتزمة الأسلاف MC إلى مجرى الدم وتهاجر إلى الأنسجة الطرفية توطين في الغالب داخل الأنسجة الضامة والأسطح الظهارية 8. وحققت النضج والتمايز النهائي في نهاية المطاف تحت تأثير سو السيتوكينات داخل الوسط 8،9 المحيطة بها. المعلقون يمكن تفعيلها من خلال مثيرة للحساسية (مستضد، حج)، الذي أدى إلى جيل من الغلوبولين المناعي E (IgE و) لقاء اكتب الأجسام المضادة. ربط هذا إيج لمستقبلات FcεRI مولودية، تليها عبر ربط الخلايا ملزمة الجلوبيولين على إعادة التعرض لنفس حج، والنتائج في FcεRI تجميع والشروع في سلسلة مما يشير إلى أن يبلغ ذروته في تحبب خلية [استعراضها في 10،11] . يتم تنشيط إم سي إس أيضا، بشكل مستقل عن فريق الخبراء الحكومي الدولي، من خلال نيوروببتيد 5،12، والسموم 13، بكتيريا ومولدات المضادات الفيروسية 14،15، وعدد من الببتيدات موجبة الشحنة يشار إليها مجتمعة ب secretagogues الأساسية، الخلايا المناعية والسيتوكينات 5،13،12،16 ، 17. يتم تنشيط المعلقون أيضا من قبل العديد من وسطاء لهم صدر الخاصة، الأمر الذي يزيد من تضخيم الاستجابة الالتهابية. معبأة المعلقون مع حبيبات إفرازية (جان الدراسات) التي تحتوي على المناعةوسطاء التنظيمية، بما في ذلك الأمينات فعال في الأوعية، مثل الهستامين والسيروتونين (في القوارض)، البروتيوغليكان، البروتياز، مثل كيماز وتريبتاز، عامل نمو بطانة الأوعية الدموية والعديد من السيتوكينات و chemokines 8،9. هذه هي وسطاء &quot;على استعداد للذهاب&quot; وحالما يتم تفعيل المعلقون من قبل التحفيز المناسب، وأصدرت هذه سطاء من الخلايا عن طريق إيماس تنظيم (تحبب) في بضع ثوان إلى دقائق 18،19. ويتبع هذا الحدث الأولي من التوليف دي نوفو وإطلاق مجموعة كبيرة من المواد الفعالة بيولوجيا، بما في ذلك الأيض حمض الأراكيدونيك، السيتوكينات متعددة وكيموكينات 20،21،22. الافراج عن المنتجات تصنيعه حديثا يحدث بشكل مستقل عن إطلاق SG. بشكل جماعي، وهذه سطاء الشروع الردود في وقت مبكر والمرحلة المتأخرة التهابات والحساسية. ولذلك، فهم آليات المحاسبة لتفعيل MC وتحبب كلاهما عفريت النظري والسريريortance. صعوبة التلاعب وراثيا إم سي إس الابتدائية ومثقف وأعاقت محاولات لتوضيح الآليات الكامنة MC تحبب، والتي ظلت حلها بشكل سيئ. للتغلب على هذه المشكلة وضعنا مقايسة على أساس مراسل بواسطة خط الخلية المخاطية الصاري transfecting المشترك، الفئران سرطان الدم مستقعد (RBL) -2H3 (المشار إليها هنا RBL) أو نخاع العظام المستمدة إم سي إس (BMMCs) 30 مع الجينات في المصالح و نيوروببتيد Y (NPY) تنصهر لRFP أحادى (mRFP)، كمراسلة SG. تم NPY توضح مسبقا تلخيص سلوك علامات SG المحلية في النظم الأخرى. وعلاوة على ذلك، لأن mRFP مضان هو الرقم الهيدروجيني حساسة والتعبير عن NPY-mRFP يسمح التصور من لجان الدراسات الحمضية وكذلك التقييم الكمي للإيماس باستخدام لوحات 96-جيدا وقارئ لوحة مضان. لقد أظهرنا أن يتم تسليم NPY-mRFP لجان الدراسات الحمضية خلايا RBL وBMMCs ويتم تحريرها من الخلايابطريقة المنظم إلى جانب البضائع SG الذاتية (أي، β-هيكسوزامينيداز والسيروتونين) 30، 32. يوفر هذا البروتوكول منهجية قائمة على التصوير عالية الدقة التي تسمح الجينات فحص الفائدة لالمظهرية وتأثيرها على خصائص وظيفية SG وتحبب في خلايا RBL 32. على وجه التحديد، وهذا البروتوكول يسمح في الوقت الحقيقي تتبع MC جان الدراسات والكمي لمنطقتهم أو حجم وحدة التخزين، عددهم، حركية التجمع والتنقل على طول الهيكل الخلوي الخلية والانصهار في نهاية المطاف مع غشاء البلازما تحت ظروف مختلفة. على سبيل المثال، توعية الخلايا مع DNP محددة إيج والتسبب في الخلايا مع حج متعددي (DNP مترافق ألبومين المصل) تحت الاضطرابات المختلفة (أي ضربة قاضية للجينات من الفائدة، على التعبير عن الجينات WT أو متحولة، أو التلاعب الدوائية) و مقارنة للسيطرة على الخلايا.

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	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Name of Material/ Equipment</td>
			<td style="width:128px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td style="width:156px;">Comments/Description</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:421px;">DMEM</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>D6046-500ML</td>
			<td style="width:156px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Fetal Bovine Serum</td>
			<td style="width:128px;">GE health care Life sciences</td>
			<td>SH30071.01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin</td>
			<td style="width:128px;">Life technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Cellulose acetate membrane, pore size 0.22&#160;&#956;m</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS430769-1EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Corning tissue-culture treated culture dishes</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS430167</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Trypsin/EDTA Solution (TE)</td>
			<td style="width:128px;">Life technologies</td>
			<td>R001100</td>
			<td style="width:156px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">PIPES dipotassium salt</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">108321-27-3&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Calcium acetate hydrate</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>114460-21-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Magnesium acetate tetrahydrate</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">M5661&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">L-Glutamic acid potassium salt monohydrate (Potassium glutamate)</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">G1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4 mm electroporation cuvettes</td>
			<td style="width:128px;">cell projects</td>
			<td style="width:139px;">EP-104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GENE PULSER WITH PULSE CONTROLLER &#38; CAPACITANCE</td>
			<td style="width:128px;">Bio rad</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chambered coverglass</td>
			<td style="width:128px;">Thermo scientific</td>
			<td align="right" style="width:139px;">155411</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">24 well, flat bottom</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS3524</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Corning 96 well plates</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS3367 or CLS390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well plate fluorescence reader- Infinite 200</td>
			<td style="width:128px;">Tecan</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium ionophore A23187</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">C7522</td>
			<td>Avoid from direct light exposure</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12-O-tetradecanoyl-13-acetate (TPA)</td>
			<td>Calbiochem</td>
			<td height="20" style="height:20px;width:139px;">P3766</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-DNP monoclonal IgE</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>D8406&#160;</td>
			<td height="20" style="height:20px;width:156px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNP-BSA/ DNP-HAS</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:139px;">A6661</td>
			<td>Avoid from direct light exposure</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Triton-x-100</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:139px;">T8787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Confocal fluorescent microscope:</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Zeiss LSM 510</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Leica</td>
			<td style="width:128px;">SP5</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nikon A1 inverted</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Imaris software</td>
			<td style="width:128px;">BITLANE</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Microsoft exel or Prism or other analyses software</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Other reagent:</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Magnesium Chloride</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">5833</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Sodium chloride</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">6404</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Calcium chloride&#160;</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">2382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin&#160;</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:139px;">A4503</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Glucose</td>
			<td style="width:128px;">BDH Laboratories</td>
			<td style="width:139px;">284515V</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Monosodium phosphate&#160;</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">5345</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Sterile water</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating mast cell secretory granules; from biosynthesis to exocytosis

الخلايا البدينة (MC) هي الخلايا الإفرازية للجهاز المناعي التي تحقق وظائفها الفسيولوجية والمرضية عن طريق الإفراج عن وسطاء الحساسية، التهابات وimmunoregulatory قبل تشكيلها وتوليفها حديثا. وسطاء إم سي إس &quot;تؤثر على الأنسجة والأعضاء متعددة بلغت ذروتها في استجابات حساسية والمناعة. وينظم درجة عالية من التوليف والتخزين والإفراج عن وسطاء MC. معبأة الوسطاء شكلت قبل في حبيبات إفرازية حشوية (SG) أن تلتحم مع غشاء البلازما والافراج عن محتواها بواسطة إيماس المنظم. نقدم بروتوكول، على أساس المشاركة في التعبير عن الجينات في المصالح مع الجين المراسل أن يتم استهداف لجان الدراسات وإصدارها بطريقة المنظم جنبا إلى جنب مع وسطاء SG الذاتية. بروتوكول يتيح عالية الدقة أربعة متحد البؤر الأبعاد يحلل من MC جان الدراسات ومراقبة الجدول الزمني الخاصة بهم من نشوء حيوي لإيماس تشغيلها. وهكذا، وذلك باستخدام هذا البروتوكول لفحص الجينات من بينمؤسسة لالمظهرية والوظيفية تأثيرها يسمح فك رموز الآليات الجزيئية التي تتحكم في نشوء حيوي وإيماس من MC جان الدراسات وتحديد الجهات الرقابية المعنية. وبالتالي، ينبغي أن تقدم مزيدا من الإيضاحات عن الآليات الخلوية التي تمثل وظيفة إم سي إس في الصحة والمرض.

ونظرا لكفاءة ترنسفكأيشن منخفضة من إم سي إس، من غير المرجح أن يترك أثرا على قراءات من متوسط ​​إفراز قياس وسطاء جان الدراسات الذاتية التلاعب الجيني. ومع ذلك، من خلال إنشاء كامل شاركت في التعبير لمراسل الجين NPY-mRFP والبلازميد-transfected شارك في نفس الخلايا، رصد النتائج NPY-mRFP ...

Watch this Scientific Journal Video about Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis at JoVE.com

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

وكان الهدف من هذا البروتوكول إلى تطوير طريقة من شأنها أن تسمح التحليلات الجينومية الوظيفية لإفراز الخلايا البدينة. ويستند هذا البروتوكول على التقييم الكمي لإطلاق سراح من الجين مراسل الفلورسنت cotrasfected مع الجينات في المصالح في الوقت الحقيقي تحليلات التشكل حبيبة إفرازية ل.

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 ...

investigating-mast-cell-secretory-granules-from-biosynthesis-to

Research

JoVE Journal

Immunology and Infection

13.0K Views.  Sackler Faculty of Medicine, Tel Aviv University.  وكان الهدف من هذا البروتوكول إلى تطوير طريقة من شأنها أن تسمح التحليلات الجينومية الوظيفية لإفراز الخلايا البدينة. ويستند هذا البروتوكول على التقييم الكمي لإطلاق سراح من الجين مراسل الفلورسنت cotrasfected مع الجينات في المصالح في الوقت الحقيق?...

Video: Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

السريرية تطبيق<em&gt; الجمال النائم</em&gt; ومستضد تقديم الخلايا الاصطناعية لتعديل وراثيا خلايا T من دم الحبل السري والمحيطية

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

عزل مجموعات فرعية B-الخلية السلائف من دم الحبل السري

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

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 ...

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon.

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

تصور الإيماس غير التحللي من<em&gt; الأدعومية المستخفية</em&gt; من الضامة الرقمية الخفيفة عن طريق الميكروسكوب

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

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

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such as skin, airways and gastrointestinal tract. Best known for their detrimental role in IgE-dependent allergic reactions, MCs have also emerged as important players in host defense against venom and invading bacteria and parasites. MC phenotype and function can be influenced by microenvironmental factors that may differ according to anatomic location and/or based on the type or stage of development of immune responses. For this reason, we and others have favored in vivo approaches over in vitro methods to gain insight into MC functions. Here, we describe methods for the generation of mouse bone marrow-derived cultured MCs (BMCMCs), their adoptive transfer into genetically MC-deficient mice, and the analysis of the numbers and distribution of adoptively transferred MCs at different anatomical sites. This method, named the &#8216;mast cell knock-in&#8217; approach, has been extensively used over the past 30 years to assess the functions of MCs and MC-derived products in vivo. We discuss the advantages and limitations of this method, in light of alternative approaches that have been developed in recent years.

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

We describe a method for the generation of in vitro derived mast cells, their engraftment into mast cell-deficient mice, and the analysis of the phenotype, numbers and distribution of engrafted mast cells at different anatomical sites. This protocol can be used to assess the functions of mast cells in vivo.

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 ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

أوفيركسريسيون وتنقية الإنسان<em&gt; رابطة الدول المستقلة</em&gt; -prenyltransferase في<em&gt; الإشريكية القولونية</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

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 ...

Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high mortality, despite new therapeutic options such as transcatheter aortic valve replacement (TAVR). 
The complete pathways that start with valve calcification and lead to severe aortic stenosis remain only partly understood. By providing a close representation of the aortic valve cells in vivo, the assaying of T lymphocytes from stenotic valve tissue could be an efficient way to clarify their role in the development of calcification. After surgical excision, the fresh aortic valve sample is dissected in small pieces and the T lymphocytes are cultured, cloned then analyzed using fluorescence activated cell sorting (FACS).
The staining procedure is simple and the stained tubes can also be fixed using 0.5% of paraformaldehyde and analyzed up to 15 days later. The results generated from the staining panel can be used to track changes in T cell concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific T cell subtypes of interest. In this study, we show the isolation of T cells, performed on fresh calcified aortic valve samples and the steps of analyzing T cell clones using flow cytometry to further understand the role of adaptive immunity in CAVD pathophysiology.

In this study, we describe the process of T lymphocyte isolation from fresh samples of calcified aortic valves and the analytical steps of T cell-cloning for the characterization of the adaptive leukocyte subsets by using flow cytometry analysis.

التحقيق في تكلس الصمام الأبهري عن طريق العزلة وثقافة الخلايا الليمفاوية T باستخدام الخلايا المغذية من معطف بافي المشعع

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high ...

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to obtain commercial monoclonal or polyclonal antibodies that recognize specific cell structures and proteins in parasites. Besides, there are few commercial antibodies available to label trypanosomatids. Usually, polyclonal antibodies against parasites are prepared in-house and could be more challenging to use in combination with other antibodies produced in the same species. Here, the protocol demonstrates how to use polyclonal and monoclonal antibodies raised in the same species to perform double labeling immunofluorescence to study host cell and pathogen interactions. To achieve the double labeling immunofluorescence, it is crucial to incubate first the mouse polyclonal antibody and then follow the incubation with the secondary mouse IgG antibody conjugated to any fluorochrome. After that, an additional blocking step is necessary to prevent any trace of the primary antibody from being recognized by the next secondary antibody. Then, a mouse monoclonal antibody and its specific IgG subclass secondary antibody conjugated to a different fluorochrome are added to the sample at the appropriate times. Additionally, it is possible to perform triple labeling immunofluorescence using a third antibody raised in a different species. Also, structures such as nuclei and actin can be stained subsequently with their specific compounds or labels. Thus, these approaches presented here can be adjusted for any cell whose sources of primary antibodies are limited.

Here, the protocol describes how to perform double labeling immunofluorescence using primary antibodies raised in the same species to study host-pathogen interactions. Also, it can include the third antibody from a different host in this protocol. This approach can be made in any cell type and pathogens.

التألق المناعي المزدوج باستخدام الأجسام المضادة من نفس النوع لدراسة التفاعلات بين المضيف والممرض

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.