Authors were funded in part by the intramural program of NIDCR (N.M.M) and supported by a Wellcome Trust Stepping Stones Fellowship (097820/Z/11/B to J.E.K) and by a Manchester Collaborative Centre for Inflammation Research grant (to J.E.K). The authors thank Teresa Wild for critically reviewing the manuscript.

وجاءت جميع الإجراءات التجريبية المبينة في هذا البروتوكول المبادئ التوجيهية اللازمة وتمت الموافقة من قبل رعاية الحيوان المؤسسية واللجنة الاستخدام، NIDCR / المعاهد الوطنية للصحة. 1. إعداد مقدما <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد وسائل الاعلام كاملة: RPMI تستكمل مع 2 مم L-الجلوتامين، و 100 وحدة / مل البنسلين، 100 ميكروغرام / مل الستربتومايسين و 10٪ FBS. </li><li style=";text-align:right;direction:rtl"> إعداد وسائل الاعلام الدناز: 50 مل من وسائل الاعلام RPMI تستكمل مع 7.5 ميكروغرام الدناز (جعل الطازجة، والحفاظ على الجليد في جميع الأوقات). </li><li style=";text-align:right;direction:rtl"> إعداد وسائل الاعلام كولاجيناز الدناز: 5 مل من وسائل الاعلام الدناز بالإضافة إلى 3.2 ملغ / مل من كولاجيناز النوع الرابع (جعل الطازجة، والحفاظ على الجليد في جميع الأوقات). </li><li style=";text-align:right;direction:rtl"> FACS العازلة قبل بارد: 0.5٪ FBS مخففة في برنامج تلفزيوني. </li><li style=";text-align:right;direction:rtl"> الطرد المركزي قبل بارد إلى 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> الدافئة شاكر حاضنة إلى 37 درجة مئوية. </li></ol> 2. عزل، تشريح، وعزل الخلايا من اللثة كتل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الموت ببطء الفئران عن طريق تعريضها إلى CO 2 في غرفة المناسبة، وفقا للمبادئ التوجيهية أراك. </li> &lt;لى&gt; فتح التجويف الصدري والبطن لفضح القلب والدورة الدموية. ليروي، إجراء شق في الوريد الأجوف السفلي ويروي 3 مل من برنامج تلفزيوني عبر البطين الأيسر باستخدام حقنة 3 مل مع إبرة 27 G. </li><li style=";text-align:right;direction:rtl"> لشل حركة الجسم والرأس من الماوس على وسادة مع المعدة مواجهة. </li><li style=";text-align:right;direction:rtl"> قطع كل صوار الشفة نحو الرقبة مع مقص، وفصل الجلد الذي يغطي الفك والعنق المنطقة، وفضح الأنسجة والعضلات الأساسية. تشريح الشفة السفلى للكشف عن الفك السفلي. </li><li style=";text-align:right;direction:rtl"> قطع ما بين القواطع السفلية لفصل جانبي الفك السفلي وشل كل جانب للوصول إلى تجويف الفم. </li><li style=";text-align:right;direction:rtl"> تصور وتشريح الحنك بعيدا عن تجويف الأنف. </li><li style=";text-align:right;direction:rtl"> تشريح المناطق باستخدام شفرة 10 وتشمل المناطق الرحى العلوية والسفلية مع توضيح اللثة (الشكل 1). جعل شقوق عمودية فقط الأمامي إلى الضرس الأول والخلفي للالضرس الثالث وشق أفقي فيحدود اللثة (ذوي الياقات البيضاء الأسنان الأنسجة المحيطة). </li><li style=";text-align:right;direction:rtl"> وضع الفك العلوي والفك السفلي كتل في 50 مل أنبوب مخروطي مع 5 مل كولاجيناز / الدناز (الحفاظ على الجليد). </li><li style=";text-align:right;direction:rtl"> احتضان في حاضنة شاكر عند 37 درجة مئوية لمدة 1 ساعة وخلال 5 دقائق الأخير من الحضانة إضافة 50 ميكرولتر من 0.5 M EDTA. </li><li style=";text-align:right;direction:rtl"> إضافة 5 مل من وسائل الاعلام الدناز البارد إلى أنبوب 50 مل مع الأنسجة ودوامة بلطف لمزج. </li><li style=";text-align:right;direction:rtl"> نقل 4 كتل من الأنسجة في طبق بتري، وتغطي مع 500 ميكرولتر من وسائل الإعلام الدناز. إزالة اللثة من كل كتلة من الأنسجة، وذلك باستخدام شفرة مشرط ولافتا نصل إلى المناطق اللثة وبين الأسنان. </li><li style=";text-align:right;direction:rtl"> نقل الأنسجة وسائل الإعلام من طبق بتري وكذلك وسائل الإعلام الدناز السابقة التي استخدمت خلال تشريح، إلى أنبوب 50 مل جديد من خلال مصفاة الخلية (70 ميكرومتر حجم). يغسل مع وسائل الإعلام الدناز (3-5 مل) كل من الأدوات المستخدمة والتصفية. </li><li style=";text-align:right;direction:rtl"> باستخدام المكبس من العقيمة حقنة 3 مل، هرس الأنسجة ضد مصفاة،تصفية مع وسائل الإعلام الدناز الباردة (30-35 مل). </li><li style=";text-align:right;direction:rtl"> غسل مصفاة الخلية مع وسائل الإعلام الدناز البارد. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي في 4 درجات مئوية، 314 x ج لمدة 6 دقائق. </li><li style=";text-align:right;direction:rtl"> تجاهل طاف، وإعادة تعليق في 1 مل من وسائل الإعلام كاملة. </li><li style=";text-align:right;direction:rtl"> عدد الخلايا (المبلغ المتوقع أن يتراوح بين 8 × 10 5-1،2 × 10 6 خلايا مع بقاء&gt; 80٪) باستخدام عداد الخلية الآلي. </li></ol> 3. تحفيز وFACS تلون خلايا اللثة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحفيز تعليق خلية واحدة مع سلطة النقد الفلسطينية (50 نانوغرام / مل)، وIonomycin (2.5 ميكروغرام / مل) في وجود 1 ميكرولتر / مل من Brefeldin أ. </li><li style=";text-align:right;direction:rtl"> احتضان لمدة 3.5 ساعة عند 37 درجة مئوية و 5٪ CO 2. </li><li style=";text-align:right;direction:rtl"> تدور باستمرار وتغسل مع برنامج تلفزيوني وأجهزة الطرد المركزي في 4 درجات مئوية. 314 x ج لمدة 6 دقائق. </li><li style=";text-align:right;direction:rtl"> وصمة عار على الخلايا باستخدام وصمة عار الجدوى باتباع إرشادات الشركة المصنعة. </li><li style=";text-align:right;direction:rtl"> وصمة عار للعلامات خارج الخلية (CD45، TCRγδ، TCRβ، CD4، CD8، TCRγδ أو نملةibodies الاختيار) باتباع إرشادات الشركة المصنعة. </li><li style=";text-align:right;direction:rtl"> غسل الخلايا مع العازلة FACS الباردة وأجهزة الطرد المركزي في 4 درجات مئوية، 314 x ج لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> دوامة بلطف لعرقلة بيليه الخلية. </li><li style=";text-align:right;direction:rtl"> إصلاح الخلايا مع بارافورمالدهيد 2٪ لمدة 20 دقيقة في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> غسل الخلايا مع العازلة FACS الباردة، الطرد المركزي في 4 درجات مئوية، 314 x ج لمدة 5 دقائق وصمة عار لالسيتوكينات داخل الخلايا. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ملء أنابيب FACS بمحلول بارد 0.5٪ سابونين المخفف في المخزن FACS وأجهزة الطرد المركزي في 4 درجات مئوية، 314 x ج لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> إضافة 300 ميكرولتر من 0.5٪ محلول سابونين للأنابيب نظام مراقبة الأصول الميدانية، واحتضان لمدة 15 دقيقة في الظلام في 4 درجة مئوية ثم الطرد المركزي في 4 درجات مئوية، 314 x ج لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> وصمة عار على الخلايا لعلامات داخل الخلايا (IL-17A وIFNγ أو السيتوكينات من خيار) باتباع إرشادات الشركة المصنعة. غسل الخلايا مع 0.5٪ محلول سابونين وأجهزة الطرد المركزي في 4 درجات مئوية، 314 x ج لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> يغسل مرة أخرى مع نظام مراقبة الأصول الميدانيةالعازلة وأجهزة الطرد المركزي في 4 درجات مئوية، 314 x ج لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> إعادة تعليق الخلايا في 300 ميكرولتر من FACS العازلة. </li></ol></li><li style=";text-align:right;direction:rtl"> تحليل النتائج على الكريات التدفق. </li></ol> 4. تحليل التدفق الخلوي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تصور الخلايا لتحليلها في الأمام (FSC) والجانب مبعثر (SCC) وبوابة لاستبعاد الحطام. </li><li style=";text-align:right;direction:rtl"> حدد الخلايا واحدة مع منطقة مبعثر إلى الأمام (FSC-A) مقابل المعلمات (FSC-H) الارتفاع. </li><li style=";text-align:right;direction:rtl"> خلايا البوابة التي يعيش (كما يتضح من تلطيخ الجدوى) وايجابية علامة CD45 + المكونة للدم لمزيد من التحليل (الشكل 2A و 2C). </li><li style=";text-align:right;direction:rtl"> مواصلة تحليل علامات محددة كما هو مبين في الشكل (2) والشكل (3). </li></ol>

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Authors have nothing to disclose

هذه التقنية الحالية غلة بنجاح عددا كبيرا من الخلايا المناعية (من ماوس واحدة) مناسبة، ليس فقط لتوصيف المظهري، ولكن أيضا للدراسات وظيفية خارج الحي. ان مجموعة اخرى تنطبق على بروتوكول لعزل وتوصيف الخلايا المناعية اللثة الفئران وعرض قيمة استخدام متعدد الألوان التدف...

أنسجة اللثة المحيطة البشرية والأسنان الفئران ويتعرضون باستمرار لبيوفيلم معقد من الأسنان 1. شبكة الخلايا المناعية الشرطة على حاجز اللثة أمر حيوي للحفاظ على سلامة الأنسجة، وضمان التوازن مع الميكروبات المتعايشة المحلية و، في نفس الوقت، وتوفير حصانة فعالة ضد التحدي الإمراض 2. لتحقيق التوازن، تم تصميم جهاز المناعة بعناية للبيئة اللثة خلق درجة عالية من التخصص، شبكة الخلايا المناعية، ولكن كما هو معروف التفاصيل الصغيرة من السكان الخلايا المناعية اللثة ودورها في الحفاظ على مناعة الأنسجة 2. عندما تتعطل توازن المناعة في اللثة، إما من خلال زيادة قابلية المضيف و / أو وجود المجتمعات الميكروبية dysbiotic، وهو حالة التهابية، اللثة تنشأ 3 4. التهاب اللثة هو مرض التهابي المشترك، مما يؤدي إلى فقدان الأسنان الصورةهياكل upporting. وينظر في أشكاله الشديدة في ما يقرب من 10٪ من عموم السكان 5. وقد ثبت تشريح العوامل الرئيسية المشاركة في حساسية اللثة والتقدم الصعب 6. ومع ذلك، فإن نماذج حيوانية مفيدة للغاية في فهم آليات بدء اللثة وتقدم 7. نماذج يمكن استخدامها لتحديد السكان الخلية الرئيسيين والوسطاء الجزيئية التي تعد حيوية للحفاظ على توازن المناعة وتطوير محرك اللثة. وهذه فكرة تحويل فهمنا لضبط اللثة محددة من توازن المناعة وتزيد من فهمنا الحالي للإمراض المرض.

<table border="0" cellpadding="0" cellspacing="0" width="914">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Fine Scissors</td>
			<td style="width:188px;">Fine science tools</td>
			<td style="width:188px;">14058-11</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Scalpel Handle #3</td>
			<td style="width:188px;">Fine science tools</td>
			<td style="width:188px;">10003-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Scalpel Blades #10</td>
			<td style="width:188px;">Fine science tools</td>
			<td style="width:188px;">10010-00</td>
			<td>Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Splinter Forceps</td>
			<td style="width:188px;">Integra Miltex</td>
			<td style="width:188px;">6-304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Needles with regular bevel&#160;</td>
			<td style="width:188px;">BD Medical</td>
			<td style="width:188px;">305109</td>
			<td>27G, 12.7 mm length</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Monoject syringes</td>
			<td style="width:188px;">Covidien</td>
			<td style="width:188px;">8881513934</td>
			<td>Luer-lock tip, 3mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">PBS, pH 7.4</td>
			<td style="width:188px;">Life Technologies</td>
			<td style="width:188px;">10010-049</td>
			<td>Without Calcium and Magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">RPMI 1640</td>
			<td style="width:188px;">Lonza</td>
			<td style="width:188px;">12-167F</td>
			<td>Without L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">DNase I from bovine pancreas</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:188px;">DN25-1G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:304px;">Collagenase type IV</td>
			<td style="width:188px;">Gibco (by Life technologies)</td>
			<td style="width:188px;">17104-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Fetal Bovine Serum</td>
			<td style="width:188px;">Gemini Bio-products</td>
			<td style="width:188px;">100-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Gentamicin 50 mg/ml</td>
			<td style="width:188px;">Quality biological</td>
			<td style="width:188px;">120-098-661EA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:304px;">Pen Strep</td>
			<td style="width:188px;">Gibco (by Life technologies)</td>
			<td style="width:188px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:304px;">L-Glutamine</td>
			<td style="width:188px;">Gibco (by Life technologies)</td>
			<td style="width:188px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">0.5M EDTA pH 8.0</td>
			<td style="width:188px;">Quality biological</td>
			<td style="width:188px;">351-027-721EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">50 mL tubes</td>
			<td style="width:188px;">Corning</td>
			<td style="width:188px;">352070</td>
			<td>Polypropylene, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">70 &#956;M Cell Strainers</td>
			<td style="width:188px;">Corning</td>
			<td style="width:188px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Petri dishes</td>
			<td style="width:188px;">Corning</td>
			<td style="width:188px;">351029</td>
			<td>Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">5 mL FACS tubes</td>
			<td style="width:188px;">Corning</td>
			<td style="width:188px;">352052</td>
			<td>Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD GolgiPlug</td>
			<td style="width:188px;">BD Biosciences</td>
			<td style="width:188px;">555029</td>
			<td>Contains brefeldin A solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Phorbol 12-Myristate 13-Acetate (PMA)</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:188px;">P8139</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Ionomycin Calcium Salt</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:188px;">13909</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Saponin from quillaja bark</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:188px;">S4521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD Fixable Aqua Dead Cell Stain Kit</td>
			<td style="width:188px;">Life Technologies</td>
			<td>L34957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD45 Alexa Fluor 700</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">56-0451-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD4 eFluor 450</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">48-0042-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse TCR beta APC eFluor 780</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">47-5961-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse gamma delta TCR FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-5711-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse IL-17A APC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">12-7311-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse IFN-&#947; PE</td>
			<td style="width:188px;">eBioscience</td>
			<td>11-5931-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse NK1.1 PE-Cy7</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">25-5941-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD90.2 APC eFluor 780</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">47-0902-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD3e FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-0031-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD19 FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-0193-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD11b FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-0112-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD11c FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-0114-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse TCR beta FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-5961-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse Ly-6G FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-5931-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse Ly-6C FITC</td>
			<td style="width:188px;">BD Pharmingen</td>
			<td style="width:188px;">553104</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, characterization and functional examination of the gingival immune cell network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

لتوضيح تطبيق البروتوكول، وتبين لنا نتائج ممثل فحص شبكة الخلايا المناعية في اللثة من الفئران مع وبدون اللثة (WT مقابل LFA - / -، الشكل 2A - C). FACS تمثيلية المؤامرات تظهر CD45 + لدم الخلايا الحية في اللثة (الشكل 2A، 2C).</s...

Watch this Scientific Journal Video about Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network at JoVE.com

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

أنشأنا تقنية لعزل وتوصيف المظهري والتحليل الوظيفي للخلايا المناعية من اللثة الفئران.

عزل وتوصيف والفحص الوظيفي لشبكة خلية اللثة المناعي

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

isolation-characterization-functional-examination-gingival-immune

Research

JoVE Journal

Immunology and Infection

10.7K Views.  NIH.  أنشأنا تقنية لعزل وتوصيف المظهري والتحليل الوظيفي للخلايا المناعية من اللثة الفئران. 

Video: Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

قياس نشاط خلايا في Calpain الثابتة والعيش من خلال التدفق الخلوي

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

عزل خلايا المناعة وظيفية القلب

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

التفريق الأدوار الوظيفية في التعبير الجيني من الخلايا المناعية وغير المناعية في التهاب القولون الماوس عن طريق زرع نخاع العظم

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

عزل تدفق Cytometric من نوع خلايا الفئران الابتدائية السنخية II طلائي للدراسات الوظيفية والجزيئية

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

العزلة، معالجة وتحليل خلايا اللثة الفئران

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

العزلة من الأنسجة الدهنية خلايا المناعة

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

ل<em&gt; في المختبر</em&gt; نموذج لقياس الاستجابات المناعية لمكافحة الملاريا في سياق الفيروس المسبب للإيدز

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

العزلة وتحليل تدفق Cytometric من الخلايا المناعية من الدماغ الدماغية ماوس

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

عزل وتوصيف المجهرية الدقيقة المستمدة من العَدلات للدراسات الفنية

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.