This work was supported by Inserm. Marion Bouvet is a fellow of the French Ministry for Research and Technology. Annie Turkieh is a fellow granted by European Union FP7 HOMAGE (305507).

بروتوكول يتبع المبادئ التوجيهية للجنة أخلاقيات البحوث البشرية مؤسساتنا. وقد تم الحصول على معطف الشهباء من المانحين البشرية السليمة من مركز نقل الدم الإقليمي (ليل الفرنسي). وأعلن عينات تم الحصول عليها من المعاطف الشهباء أنها مجموعة INSERM (رقم DC2010-1209). 1. المواد والثقافة تحضير الوسائط <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تمييع 10X الفوسفات عازلة المالحة (PBS) في الماء المقطر المعقم للحصول 1X PBS. </li><li style=";text-align:right;direction:rtl"> جعل المتوسط ​​1640 RPMI تستكمل مع الجنتاميسين (40 ميكروغرام / مل) ولام الجلوتامين (2 ملم)، مع وبدون 10٪ تجميع المصل البشري. </li></ol> 2. الثقافات الأولية من البلاعم المشتقة من الوحيدات (MDM) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تمييع buffycoat (25 مل) مع 1X PBS. تحميل بعناية على Ficoll / leucosep وأنبوب الطرد المركزي في 1600 x ج لمدة 20 دقيقة في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> جمع وحيدات في واجهة في أنبوب جديد. غسل 3X مع 10 مل 1X PBS تحتوي على 0.1٪ ethylenediaminetetraحمض الخليك (EDTA) من خلال centrifugations التوالي في 1000 x ج، 370 x ج و 160 x ج لمدة 10 دقيقة لكل منهما، ثم مرة واحدة في برنامج تلفزيوني 1X وحدها في 160 x ج لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> resuspend الكرية خلية في المتوسط ​​5 مل RPMI-1640 دون المصل والبذور الخلايا في أطباق 35 مم في مناطق ذات كثافة من 1 × 10 6 خلايا في طبق. </li><li style=";text-align:right;direction:rtl"> بعد الترسيب لمدة 90 دقيقة في الحاضنة، تجاهل طاف تحتوي الخلايا غير ملتصقة. غسل الخلايا الملتصقة، وتتألف من وحيدات، 3X مع 1 مل PBS. ثم يضاف 1 مل من المتوسط ​​الطازجة تحتوي على 10٪ (V / V) في مصل الدم البشري إلى خلايا حرة المصل سابقا. </li><li style=";text-align:right;direction:rtl"> بعد 6 أيام من الثقافة، لتسفر الضامة متباينة البديلة (M2)، إضافة المؤتلف الإنسان IL-4 (15 نانوغرام / مل)، والحفاظ على لمدة 6 أيام. ثم، علاج الضامة متباينة مع عديد السكاريد الشحمي (100 نانوغرام / مل) في اليوم 12 لمدة 4 ساعة للحصول الضامة M1. </li></ol> 3. استخراج M1 و M2 بلعم البروتينات ل2D الكهربائي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> غسل macrophالأعمار ثلاث مرات مع 25 ملي تريس، ودرجة الحموضة 7.4، والخردة في العازلة التي تحتوي على 30 ملي تريس درجة الحموضة 8، 4٪ 3 - [dimethylammonio (3-cholamidopropyl)] -1-propanesulfonate (الفصول)، 2 M ثيوريا و 7 M اليوريا. </li><li style=";text-align:right;direction:rtl"> ليز خلايا باستخدام خلاط مناسبة ل1.5 مل أنابيب microcentrifuge لمدة 5 دقائق في الثلج ومخزن في -20 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> تحديد تركيز البروتين باستخدام كاشف برادفورد التجاري. تخزين 100 مكل من البروتينات في -20 درجة مئوية حتى الاستخدام. </li></ol> 4. Isoelectrofocusing ملاحظة: تنفيذ جميع إجراءات وضع العلامات في الظلام. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تخفيض 5 ميكروغرام لكل عينة (M1 و M2 الضامة) تعدل إلى 9 ميكرولتر مع العازلة تحلل مع 2 ملي تريس (2-carboxyethyl) الفوسفين (TCEP) لمدة 1 ساعة عند 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> إضافة cyanine 3 (و Cy3) لاستخراج M1 و M2-سلفهيدريل رد الفعل استخراج صبغة بتركيز 0.8 نانومتر البروتين / ميكروغرام. إضافة cyanine 5 (Cy5) إلى M1 و M2 مقتطفات بتركيز 0.8 نانومتر / ميكروغرام من البروتين واحتضانلمدة 30 دقيقة عند 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> وقف رد الفعل مع إضافة حجم مساو من العازلة العينة تحتوي على 7 M اليوريا و 2 M ثيوريا، CHAPS٪ 4، 130 ملي dithiothreitol (DTT) و 2٪ pharmalytes. </li><li style=";text-align:right;direction:rtl"> مزيج و Cy3 المسمى العينات مع عينات M1 M2 Cy5 المسمى في يد واحدة و Cy3 المسمى عينات M2 مع Cy5 المسمى عينات M1 في جهة أخرى. </li><li style=";text-align:right;direction:rtl"> ترطيب التدرج الحموضة مشلول (IPG) شريط (240 ملم، ودرجة الحموضة 3-10 الانحدار الخطي) مع 450 ميكرولتر من عينات مختلطة وصفت في العازلة التي تحتوي على 7 M اليوريا و 2 M ثيوريا و 4٪ CHAPS على بأر متساوي التكهرب (منتدى الطاقة الدولي) نظام خلية لمدة 24 ساعة دون تطبيق أي تيار. </li><li style=";text-align:right;direction:rtl"> أداء التركيز على 300 فولت (V) لمدة 3 ساعة، وبعد ذلك في الانحدار إلى 1000 V لمدة 6 ساعة، في التدرج إلى 8000 V لمدة 3 ساعة وأخيرا في 8000 V لمدة 3 ساعة. </li></ol> 5. البعد الثاني ملاحظة: تنفيذ جميع الإجراءات الكهربائي في الظلام. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> احتضان شرائط IPG العازلة في موازنة تحتوي على0.1 ملي تريس، حمض الهيدروكلوريك (الرقم الهيدروجيني 8)، 6 M اليوريا و 2٪ (ث / ت) كبريتات الصوديوم دوديسيل (SDS) و 30٪ (V / V) الجلسرين جي لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> نقل شرائط IPG معايرتها عن البعد الثاني (SDS-إس دي إس بايج (PAGE)) على 12.5٪ والمواد الهلامية PAGE وختم مع انخفاض ذوبان الاغاروز. </li><li style=";text-align:right;direction:rtl"> تنفيذ الكهربي عند 20 درجة مئوية باستخدام نظام Ettan-Daltsix في الجهد المستمر 70 فولت تبع بين عشية وضحاها بنسبة 300 V حتى تصل الجبهة الزرقاء برموفينول الجزء السفلي من هلام. </li></ol> 6. الحصول على الصور وتحليل بيوينفورمتيك <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> المواد الهلامية مسح يلقي بين اثنين من لوحات الزجاج المنخفض مضان مع ماسح ضوئي DIGE تصوير في موجات الإثارة / انبعاث 532/580 نانومتر لو Cy3 633/670 نانومتر لCy5 لانتاج صور ذات حجم بكسل 100 ميكرون. </li><li style=";text-align:right;direction:rtl"> إجراء تحليل الصور مع البرمجيات التجارية كما هو موضح سابقا (12). </li><li style=";text-align:right;direction:rtl"> حساب وتطبيع حجم البقعة في كل صورة. تعيين حجم البقعة تطبيع كدعامةortion من القيمة الإجمالية لكل بقعة اكتشفت في هلام. </li><li style=";text-align:right;direction:rtl"> تحليل الاختلافات في حجم البقعة البروتين لكل نوع من أنواع الضامة بمقارنة قيمة حجم بقعة طبيعية بين المجموعتين (M1 و M2). النظر في الفرق بين حجم البقعة لتكون كبيرة إذا كان التغيير هو 1.5 أضعاف (P &lt;0.05، في اتجاه واحد تحليل أنوفا). </li></ol>

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There are no declared conflict of interest.

بروتوكول الموصوفة هنا تفاصيل طريقة لتحليل تأثير محفزات مختلفة من اثنين من الأنواع الفرعية الضامة، M1 (الموالية للالتهابات) و M2 (المضادة للالتهابات). وقد تم الحصول على الثقافات الأولية من M1 و M2 الضامة من التفريق بين حيدات كما نشرت سابقا 7. <p class="jove_content" style=";text-align...

الضامة هي خلايا غير متجانسة والبلاستيك التي تكون قادرة على الحصول على الظواهر وظيفية متميزة. وفي الجسم الحي، وهذه الخلايا تستجيب لمجموعة كبيرة ومتنوعة من signalssuch البيئية الدقيقة عن المنتجات الميكروبية، السيتوكينات، الخ. 1. في المختبر، والنمط الظاهري الموالية للالتهابات (M1) من البلاعم يمكن أن يسببها عديد السكاريد الشحمي (LPS) والنمط الظاهري المضادة للالتهابات (M2) من خلال بعض السيتوكينات مثل انترلوكين 4 (IL-4). وعلاوة على ذلك، يمكن الضامة التبديل من M1 M2 النمط الظاهري لتفعيلها، وعلى العكس، بناء على إشارات معينة 2. اعتمادا على النمط الظاهري، سوف الضامة لها وظائف مختلفة. الضامة M1 هي الخلايا التي تنتج السيتوكينات الموالية للالتهابات، مثل عامل نخر الورم α (TNF-α)، لقتل الميكروبات أو الخلايا السرطانية 3. في المقابل، M2 الضامة تمنع هذه الاستجابة الالتهابية مثل في التئام الجروح والتليف عن طريق إنتاج مكافحة inflammatorذ العوامل مثل TGF-β 3،4. تم عزل الخلايا الدموية المحيطية وحيدات النوى الإنسان من المانحين صحية من خلال التدرج Ficoll الكثافة الطرد المركزي كما هو موضح سابقا 5 باستخدام تقنية مقتبسة من Boyum 6. يمكن التفريق بين الضامة في الثقافة إلى M1 أو M2 النمط الظاهري بعد 6 أيام من الثقافة الابتدائية 7. تحليل تعبير البروتين أو البروتين تغييرات بين اثنين من الأنواع الفرعية الضامة تحت مؤثرات تسيطر مختلفة، مثل المرضية المضيف أو السموم الميكروبية، وسوف تكون مفيدة فك وظائف المؤيدين والضامة المضادة للالتهابات. البروتينات هي أدوات فريدة من نوعها لرصد مباشر من البروتينات التي هي حتى- على وجه التحديد أو التنظيم إلى أسفل في الضامة الإنسان المثقف في إطار مختلف المنبهات. وقد حل الأصباغ الفلورية بعض القيود من 2D الكهربائي للهلام، مثل انخفاض الحساسية وتحليل الصور 8 &lt;<html> / سوب&gt;. الأصباغ التفاعل مع بقايا السيستين زادت حساسية الكشف مقارنة مع أولئك التفاعل مع بقايا يسين 9. في دراسة سابقة، أثبتنا فائدة DIGE الوسم تشبع لتحليل العينات النادرة 10 مقارنة الملطخة الفضة الكلاسيكية 2D الكهربائي 11. هذه التقنية مفيدة في تحليل سريع التعديلات البروتين بين اثنين من الأنواع الفرعية الضامة أو بين الضامة غير المعالجة والمعالجة من نفس النوع الفرعي. مزايا هذه التقنية هي وجود البروتين الحصول على المعلومات من حجم البروتين والتعديلات بعد متعدية من خلال تحليل هلام 2D 12. يجب أن يؤخذ في الاعتبار أن هذه ليست تقنية إنتاجية عالية، مما يحد من عدد العينات التي يمكن تحليلها. تطوير فحوصات إنتاجية عالية تعتمد على الطيف الكتلي كما استعرضت مؤخرا 13 يمكن أن يحسن هذا. _content &quot;&gt; هنا نقدم كيفية إجراء تحليل 2D DIGE من استخراج البروتين الضامة مثقف من خلال عمليات الكهربائي، isoelectrofocusing، وSDS-PAGE وكذلك معلومات عن فائدة برنامج 2D كاف.

<table border="0" cellpadding="0" cellspacing="0" width="472">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:208px;"><a name="RANGE!A1:C43">Name</a></td>
			<td style="width:155px;">Company</td>
			<td style="width:111px;">Catalog number</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RPMI 1640</td>
			<td>Invitrogen</td>
			<td>31870-074</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PBX 10 X</td>
			<td>Invitrogen</td>
			<td>14200-083</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">L-glutamine-200mM-100X</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">gentamycin 10 mg/mL</td>
			<td>Invitrogen</td>
			<td>15710-049</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">human serum</td>
			<td>Invitrogen</td>
			<td>34005100</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ficoll d=1,077</td>
			<td>ATGC</td>
			<td>L6115</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Leucosep</td>
			<td>Dutscher</td>
			<td>16760</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#160;6-wells plate PRIMARIA&#160;</td>
			<td>Becton Dickinson</td>
			<td>353846</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IL-4</td>
			<td>Promocell</td>
			<td>B-61410</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">lipopolysaccharide</td>
			<td>Sigma-Aldrich</td>
			<td>L-2654</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Giemsa</td>
			<td>Fluka</td>
			<td>48900</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">EDTA MM372,2</td>
			<td>Research Organics&#160;</td>
			<td>3.00E+01</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Filter 0,22&#181;m</td>
			<td>Millipore</td>
			<td>SCGPTORE</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">100 mL cylinder</td>
			<td>Corning</td>
			<td>430182</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TCEP</td>
			<td>Interchim</td>
			<td>UP242214</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bradford reagent</td>
			<td>Bio-Rad</td>
			<td>5000006</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cy3+Cy5-reactive dye</td>
			<td>GE Healthcare</td>
			<td>25-8009-83</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IPG strip 3-10 24cm</td>
			<td>GE Healthcare</td>
			<td>17-6002-44</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Protean IEF cell</td>
			<td>Bio-Rad</td>
			<td>165-4000</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Low-melting agarose</td>
			<td>Invitrogen</td>
			<td>15517-014</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ettan-Daltsix system</td>
			<td>GE Healthcare</td>
			<td>80-6485-08</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ettan DIGE Imager scanner</td>
			<td>GE Healthcare</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Progenesis Samespot</td>
			<td>Non linear dynamics</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">50 ml tubes</td>
			<td>any supplier</td>
			<td>n/a</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">15 ml tubes</td>
			<td>any supplier</td>
			<td>n/a</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">CHAPS</td>
			<td>Sigma-Aldrich</td>
			<td>C5070</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ur&#233;e</td>
			<td>Merk</td>
			<td>108484-500</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Thiour&#233;e</td>
			<td>Sigma-Aldrich</td>
			<td>T7875</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DTT</td>
			<td>Bio-Rad</td>
			<td>1610611</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">APS</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TEMED</td>
			<td>Sigma-Aldrich</td>
			<td>T9281</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tris Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tris HCl</td>
			<td>Sigma-Aldrich</td>
			<td>T3253</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pharmalytes 3-10</td>
			<td>GE Healthcare</td>
			<td>17-0456-01</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">SDS</td>
			<td>Sigma-Aldrich</td>
			<td>L3773</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>114391</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G6279</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Acrylamide 40%</td>
			<td>Bio-Rad</td>
			<td>161-0148</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">2D clean Up</td>
			<td>GE Healthcare</td>
			<td>80-6454-51</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dim&#233;thylformamide</td>
			<td>Sigma-Aldrich</td>
			<td>22705-6</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">electrode wicks</td>
			<td>Bio-Rad</td>
			<td>165-4071</td>
		</tr>
	</tbody>
</table>

proteomic profiling of macrophages by 2d electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

لأداء التفاضلية تحليل البروتين الملائم، يجب التأكد من صحتها تجهيز عينات ليتم تحليلها. في المثال المقدمة، مطلوب نوعية زراعة الخلايا الضامة للجوانب الشكلية والجزيئية كما نشرت سابقا 5. وأعقب تمايز حيدات في الضامة وتجانس الثق...

Watch this Scientific Journal Video about Proteomic Profiling of Macrophages by 2D Electrophoresis at JoVE.com

Proteomic Profiling of Macrophages by 2D Electrophoresis

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

البروتين من التنميط الضامة التي كتبها 2D الكهربائي

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

proteomic-profiling-of-macrophages-by-2d-electrophoresis

Research

JoVE Journal

Immunology and Infection

12.6K Views.  University Lille Nord de France. Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

Video: Proteomic Profiling of Macrophages by 2D Electrophoresis

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

نضوب وإعادة إعماره الضامة في الفئران

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

2D و 3D كروموسوم اللوحة في البعوض الملاريا

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

دراسة يحلول يبلوعي النشوء الأحيائي في الضامة لايف

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

استخدام<em&gt; الشيجلا الفلكسنرية</em&gt; لدراسة التفاعلات الالتهام الذاتي الهيكل الخلوي

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

تقييم نشاط مضاد للفطريات المعزولة من السنخية الضامة التي كتبها متحد البؤر المجهري

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

ترنسفكأيشن كفاءة عالية من الإنسان THP-1 الضامة بواسطة Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

الكمي عصاري خلوي مقابل رغوي<em&gt; السالمونيلا</em&gt; في الضامة الابتدائية التي كتبها التفاضلية Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

التوصيف المظهري لالضامة من الكلى الجرذ بواسطة التدفق الخلوي

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.

SILAC القائم [بروتيوميك توصيف Exosomes من HIV-1 الخلايا المصابة

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

البروتين من التنميط الضامة التي كتبها 2D الكهربائي

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