وأيد NGL من قبل مبادرة جامعة واين ستيت في فترة ما حول الولادة صحة الأم وفترة ما حول الولادة وصحة الطفل. نحن نعترف بامتنان مورين McGerty وايمي E. Furcron (جامعة ولاية واين) لقراءة نقدية لها من المخطوطة.

الكتاب تكشف عن أي تضارب في المصالح.

جمع البيانات متسقة أن يسجل وفرة والمظهرية خصائص الكريات البيض التسلل في واجهة الأم والجنين ضروري لفهم التسبب في مضاعفات مرتبطة بالحمل. وقد وصفت العديد من التقنيات التي تسهل عزل التسلل الكريات البيض من أنسجة الفئران في واجهة الأم والجنين طوال فترة الحمل 31،38،39،43-46....

التسامح المناعي في الحمل هي فترة عندما تحدث تغيرات مميزة في الجهاز المناعي للأم. هذه التغييرات تسمح الأم لتحمل الجنين، والكسب غير المشروع شبه الصنعية 1. الجنين يعبر عن الأب معقد التوافق النسيجي الرئيسي (MHC) المستضدات 2، وقد تم العثور على خلايا الجنين في الدورة الدموية للأم 3. ومع ذلك، لا يتم رفض الجنين 4،5. ليست مفهومة تماما هذا اللغز. تنص الفرضية الأخيرة أن التسامح الأم والجنين يتم إنشاؤه أثناء الجماع وتلقيح 6،7 وصيانتها للحفاظ على الحمل فترة ولاية كاملة 10/08. ويعتبر توزيع لهذا التسامح الأم والجنين آلية المرض خلال المراحل المبكرة والمتأخرة من الحمل 10-16. التسامح الأم والجنين ينطوي على مشاركة مختلف المجموعات الفرعية من السكان الكريات البيض، بما في ذلك خلايا T (خلايا T التنظيمي، وخلايا TH1 الخلايا TH2، وخلايا Th17)، لجنة الهدنة العسكريةrophages، العدلات، الخلايا البدينة، الخلايا القاتلة الطبيعية، والخلايا NKT، والخلايا الجذعية، والخلايا B، أن التغير في كثافة وتوطين طوال فترة الحمل 15،17-19. غير المخصب التسامح الأم والجنين على الأم والجنين واجهة 20 - الموقع التشريحي حيث يتفاعل الجهاز المناعي للأم مع الجنين مستضدات 20،21. يتم إنشاء واجهة الأم والجنين خلال المشيمة عندما خلايا الأرومة الغاذية extravillous الجنين تغزو الغشاء المخاطي في الرحم 22-24. على الجانب الجنين من هذه الواجهة، والأغشية المحيطة بالجنين إنشاء سطح الظهارية المتخصصة في المشيمة، وخلايا الأرومة الغاذية المخلوية السيطرة على صرف المواد الغذائية من خلال الاتصال المباشر مع دم الأم (22). على الجانب الأمهات واجهة، والساقط المجندين تجمع غير متجانس من الكريات البيض التي في الفئران تمثل 30٪ إلى 50٪ من جميع الخلايا الساقطية. بالإضافة إلى مشاركتهم في maternآل التسامح المناعي، وهذه الخلايا هي من أهم المساهمين في عمليات مختلفة خلال فترة الحمل، على سبيل المثال، حماية الجهاز التناسلي من الالتهابات والتلقيح، زرع الجنين 7،25، الأوعية الدموية ساقطي 26، إعادة الأوعية الدموية 24،27، غزو الأرومة الغاذية 28، المشيمة التنمية 24،25، وفي نهاية المطاف والمخاض والولادة 15،17. ولذلك، فإن دراسة الكريات البيض تشارك في التسامح الأم والجنين ضروري لتوضيح التسبب في مضاعفات مرتبطة بالحمل. في حين أن استخدام المناعية والمناعي ولدت البيانات لرؤية مباشرة وتوطين الرحم، ساقطي، أو الكريات البيض المشيمة 29،30، تحليل التدفق الخلوي وكذلك كشفت مجموعات فرعية محددة من الكريات البيض في كل من هذه الأنسجة 31،32. بالإضافة إلى ذلك، التدفق الخلوي قد استخدمت لتحديد الكثافة ونسبة الأمنال والجنين واجهة الكريات البيض 33 و التعبير مستويات بروتينات الخلية والخلايا 8-10،34. تحليل تدفق cytometric من الكريات البيض في واجهة الأم والجنين يتطلب تعليق وحيد الخلية. من أجل عزل الكريات البيض التسلل من ساقطي، الرحم، والأنسجة المشيمية، وقد استخدمت طريقتين للتفكك النسيج: الميكانيكية والأنزيمية. كلتا الطريقتين تسمح للفصل الكريات البيض تسللوا من المصفوفة خارج الخلية (ECM) من هذه الأنسجة. تفكك النسيج الأنزيمية متفوقة على تفكك النسيج الميكانيكية لأنها تسمح عائدات أعلى من الكريات البيض مع الأضرار المرتبطة القص قوة أقل 35. ونتيجة لذلك، تفارق الأنسجة الميكانيكية يتطلب إلى التجميع الأنسجة 36، والتي قد تزيد من التباين والتغاير من العينات. بعد ذلك، قد يكون التفكك الميكانيكية الخيار عندما يكون المستضد من الفائدة يمكن أن تتغير من التفكك الأنزيمية أو عند وظيفة الخليةأن الحفاظ على الصورة الحاجة الفائدة (على سبيل المثال، السمية الخلوية للخلايا NK) 35. استخدام التحلل البروتيني مع إنزيمات معينة لتدهور ECM يلغي العائدات المنخفضة لاحظ مع التفكك الميكانيكية. وأفادت العديد من الدراسات أن استخدام التربسين 32، كولاجيناز 37، الدناز 31، dispase 38، والكوكتيلات التجارية من الإنزيمات المختلفة 32،39. ومع ذلك، فإن طبيعة وتركيز الإنزيمات المختلفة ومدة الهضم يجب تحديدها بدقة والتأكد من صلاحيتها لضمان الحفاظ على سلامة الحواتم المستضدية سطح الخلية المطلوبة لالمناعي. الهياكل سطح مختلف عرضة بشكل مختلف لتدمير الإنزيمات المختلفة، مع بعض الإنزيمات، مثل التربسين، كونها سيئة السمعة لتجريد الحواتم سطح الكريات البيض معترف بها من قبل العديد من الأجسام المضادة وحيدة النسيلة. قدم هنا هو طريقة استخدام proteolytجيم وكوكتيل الأنزيمية حال للكولاجين، ودعا Accutase. هذا الحل الأنزيمية هو لطيف بما فيه الكفاية في حين لا يزال فعالا في الفصل بين أنسجة الفئران في واجهة الأم والجنين، و لا يتطلب إضافة الكواشف النأي أخرى أو مصل لإنهاء رد فعل التفكك. وعلاوة على ذلك، وانها مستعدة لاستخدام الموردة و، على الرغم من أن الوقت من التفكك يحتاج إلى التحقق، وهو أكثر قوة من الانزيمات المشار إليها أعلاه 40،41. استخدام مزيج من كلا النوعين من تصنيف الأنسجة يحسن نوعية وكمية من الخلايا التي تم الحصول عليها. وبالتالي، العديد من الدراسات قد نفذت الجمع بين استخدام تفارق الميكانيكية والأنزيمية مع نتائج مرضية 31،32،37. تأسست بروتوكول صفها هنا والتحقق من صحتها في مختبرنا. يستخدم مزيج من التفكك الميكانيكية لطيف تليها تصنيف الأنزيمية القوي. يسمح هذا البروتوكول الدراسة العزلة ومزيد منالكريات البيض التسلل في أنسجة الفئران في واجهة الأم والجنين (الرحم، الساقط، والمشيمة). يحافظ على بروتوكول التالية أيضا على سلامة علامات سطح الخلية وعوائد خلايا قابلة للحياة بما فيه الكفاية لتطبيقات المصب كما يتبين من تحليل تدفق cytometric. أخيرا، ويحافظ على هذا البروتوكول اتساق إعداد الخلايا لتحليل ومقارنة أنسجة الفئران مختلفة تتكون واجهة الأم والجنين.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Magentic Cell Separation</td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Separator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30&#956;m pre separation filters</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multistand</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15mL safe lock conical tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Buffer</td>
			<td></td>
			<td></td>
			<td>(0.5% bovine serum albumin, 2mM EDTA and 1X PBS)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Anti-mouse CD16/CD32</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse extracellular antibodies</td>
			<td></td>
			<td></td>
			<td>(Table 1)</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td></td>
			<td></td>
			<td>(BSA)</td>
		</tr>
		<tr>
			<td>LIVE/DEAD viability dye</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fixation buffer solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Buffer</td>
			<td></td>
			<td></td>
			<td>(1% bovine serum albumin, 0.5% sodium azide, and 1X PBS ph 7.2)</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution 0.4%</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Additional Instruments</td>
		</tr>
		<tr>
			<td>Incubator with shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of leukocytes from the murine tissues at the maternal-fetal interface

التسامح المناعي في الحمل يتطلب أن النظام المناعي للأم يخضع لتغيرات مميزة من أجل قبول ورعاية الجنين. يبدأ هذا التسامح أثناء الجماع، أنشأ خلال تلقيح والزرع، وحافظت طوال فترة الحمل. يتم إثراء وسطاء الخلوية والجزيئية النشطة التسامح الأم والجنين في موقع التماس بين أنسجة الجنين والأم، والمعروفة باسم واجهة الأم والجنين، والذي يتضمن المشيمة والرحم والأنسجة الساقطية. وتتألف هذه الواجهة من خلايا انسجة والكريات البيض التسلل، ووفرة والمظهرية خصائصها تتغير على مدى فترة الحمل. وتشمل التسلل الكريات البيض في واجهة الأم والجنين العدلات، الضامة، والخلايا الجذعية، الخلايا البدينة، وخلايا T والخلايا B والخلايا القاتلة الطبيعية، والخلايا NKT التي تخلق معا المحلية الصغيرة للبيئة التي تحافظ على الحمل. اختلال التوازن بين هذه الخلايا أو أي inappropيعتبر تغيير riate في الظواهر التي آلية المرض في فترة الحمل. ولذلك، فإن دراسة الكريات البيض التي تتسلل إلى واجهة الأم والجنين أمر ضروري من أجل توضيح الآليات المناعية التي تؤدي إلى مضاعفات متعلقة بالحمل. الموصوفة هنا هو البروتوكول الذي يستخدم مزيجا من التفكك الميكانيكية لطيف تليها تصنيف الأنزيمية قوية مع بروتين وحال للكولاجين كوكتيل الأنزيمية لعزل الكريات البيض التسلل من أنسجة الفئران في واجهة الأم والجنين. هذا البروتوكول يسمح لعزل أعداد كبيرة من الكريات البيض قادرة على البقاء (&gt; 70٪) مع خصائص المستضدات وظيفية الحفظ بما فيه الكفاية. ومن ثم يمكن تحليلها الكريات البيض معزولة من قبل العديد من التقنيات، بما في ذلك المناعي، فرز الخلايا، والتصوير، immunoblotting، مرنا التعبير، زراعة الخلايا، وفحوصات في المختبر وظيفية مثل ردود فعل متباينة الكريات البيض، والانتشار، أو فحوصات السمية الخلوية.

يظهر تشريح الأنسجة الفئران من واجهة الأم والجنين في الشكل 1؛ ويشمل هذا الإجراء فتح التجويف البريتوني (الشكل 1A، B)، وأبواق الرحم (الشكل 1C) بما في ذلك مواقع زرع (الشكل 1D)، وجمع من أنسجة الرحم (الشكل 1E)، المشيمة (الشكل 1F)،</str...

Watch this Scientific Journal Video about Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface at JoVE.com

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

عزل الكريات البيض من أنسجة الفئران في واجهة الأم والجنين

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

isolation-leukocytes-from-murine-tissues-at-maternal-fetal

Research

JoVE Journal

Immunology and Infection

17.1K Views.  Wayne State University School of Medicine. Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis. 

Video: Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

عزل الكريات البيضاء في الدماغ التسلل

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

عزل الحمض النووي الريبي الزائفة الزنجارية استعمار الهضمي المسالك الفئران

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

العزلة وتحليل الدماغ المعزول من الكريات البيضاء<em&gt; المتصورة berghei</em&gt; ANKA الفئران المصابة

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

الطاقة الحيوية والأكسدة انفجار: بروتوكولات لعزل وتقييم الكريات البيضاء والصفائح الدموية البشرية

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

عزل حيدات الإنسان من مزدوجة التدرج الطرد المركزي والتمايز لالضامة في تفلون المغلفة حقائب خلية ثقافة

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

تحليل آثار خلايا انسجة على تجنيد الكريات البيض من التدفق

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

العزل وزرع مختلفة المسنين الفئران التوتة الطعوم.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

عزل الكريات البيضاء من واجهة الإنسان الأم والجنين

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

عزل التسلل الكريات البيض من ماوس الجلد عن طريق الأنزيمية دايجست والفصل التدرج

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

الثقافات المشيمة وساقطي الجهاز الإنسان لدراسة العدوى في واجهة الأم والجنين

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...