Name: characterization of human monocyte-derived dendritic cells by imaging flow cytometry: a comparison between two monocyte isolation protocols
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This research is supported by the National Institute on Alcohol Abuse and Alcoholism, award K99/R00 AA021264. Additional lab support as part of startup package has been received from the Department of Immunology, Institute of NeuroImmune Pharmacology, Herbert Wertheim College of Medicine, and FIU- Office of Research and Economic Development. Gianna Casteleiro was supported by NIH/NIGMS R25 GM061347. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

وقد تم استعراض الدراسات الدم البشرية الشاملة والموافقة عليها من قبل مجلس المراجعة المؤسسية (IRB) من وحدة الاستخبارات المالية، بروتوكول الاتحاد الدولي للرجبي موافقة الاتحاد الدولي للرجبي # 13-0440. تم شراؤها leukopaks الإنسان من بنك الدم المجتمع في ميامي، فلوريدا. <p class="jove_ti...

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وبناء على الصعوبات المعروفة للعزل وتوليد MDDCs من دم الإنسان، هذه الدراسة تهدف إلى تقديم مقارنة شاملة طريقتين راسخة لتوليد MDDCs. الطريقة الأولى مقارنة هو الطريقة التقليدية الراسخة لتوليد MDDCs من خلال استغلال قدرة حيدات التمسك الزجاج أو البلاستيك (طريقة الالتزام) 21،22،2...

الخلايا الجذعية (DCS) هي سطاء الأساسية للأنظمة المناعة الفطرية والتكيفية. أنها تعمل على إحداث الاستجابة المناعية الأولية وتسهيل تطوير الذاكرة المناعية. هذه الخلايا هي المسؤولة في المقام الأول عن القبض على مستضد والهجرة وتحفيز الخلايا التائية، وبالتالي يشار إلى مستضد الخلايا كما المهنية (ناقلات الجنود المدرعة) 1 .Manipulation من البلدان النامية يمكن استخدامها عبر مجموعة واسعة من المجالات البحثية وفي إعداد سريرية لعلاج مختلفة الأمراض الالتهابية مثل فيروس نقص المناعة البشرية 6،7، سرطان 8، 9 أمراض المناعة الذاتية، والاستجابات التحسسية 10. كما تستخدم البلدان النامية للبحث تعاطي المخدرات من أجل حل آليات ومسارات مجهولة مثل تلك المرتبطة إدمان الكحول 11-14، إدمان المخدرات 13،15، والجمع بين العدوى وتعاطي المخدرات فيروس نقص المناعة البشرية 16-19. هذه الدراسات الجارية والدراسات البحثية المستقبلية طن مجال علم المناعة تجعل في الجيل المختبر من البلدان النامية في غاية الأهمية للبحث. ومع ذلك، هناك العديد من الصعوبات المرتبطة عزل البلدان النامية من الدم البشري لأنها تشكل فقط 0،1-1٪ من مجموع خلايا الدم وحيدات النوى 20. حتى الآن، بعض الطرق الراسخة لتوليد البلدان النامية في المختبر يتكون من البلاستيك أو الزجاج التزام حيدات 21،22، والكثافة الطرد المركزي التدرج 23، محددا الفصل على علامة مثل خلية تنشيط المغناطيسية فرز 22، فلوري خلية تنشيط الفرز 24، واختيار إيجابية من وحيدات CD14 + باستخدام المغلفة ديكستران النانوية المغناطيسية 25، والعزلة السريع لحيدات العالية النقاء باستخدام مؤتمتة بالكامل السلبية تحديد الخلية 26. ومع ذلك، فإن أفضل طريقة لاختيار ما زال مثيرا للجدل. لذلك، لتحسين تقنيات الجيل العاصمة، وقد وضعت العديد من الطرق التينقاء هذه الخلايا يمكن أن يزيد بشكل كبير من التمايز من CD34 + السلف الخلايا وحيدات تنقية معزولة من خلايا الدم وحيدات النوى المحيطية (PBMCs) 27. كما ذكر قبل، وهي طريقة تستخدم على نطاق واسع وشعبية لتوليد الوحيدات المستمدة الخلايا الجذعية (MDDCs) هو استكشاف قدرة حيدات التمسك الزجاج أو البلاستيك (طريقة الالتزام) 21،22،27. طريقة الالتزام هو طريقة سريعة ومباشرة التي لا تتطلب استخدام معدات معقدة. ومع ذلك، بعض مساوئ هذه العملية تشمل تلوث اللمفاويات، مرونة منخفضة، والوحيدات التلاعب عابر 28. طريقة بديلة لطريقة الالتزام هو العزلة المغناطيسية للحيدات من إجمالي PBMCs، خصوصا مع استخدام عدة تخصيب الوحيدات الإنسان، الذي يهدف إلى عزل حيدات من PBMCs عن طريق الانتقاء السلبية 26. خلال هذا الإجراء، يتم استهداف الخلايا غير المرغوبة لإزالة مع رباعي القسيماتمجمعات الأجسام المضادة والجسيمات المغناطيسية المغلفة ديكستران. وميزة هذه الطريقة العزلة هي أن الخلايا المسمى غير المرغوب فيها يتم فصل باستخدام مغناطيس في حين أن الخلايا المستهدفة يمكن سكب بحرية قبالة في أنبوب جديد دون الحاجة للأعمدة. حتى الآن، مع توافر الاجسام المضادة المحددة التي يمكن تسمية السكان الخلية فريدة من نوعها، أصبحت تقنية الفصل المغناطيسي ليس مجرد وسيلة إضافية، بل ضرورة لعزل خلايا نادرة في مجال علم المناعة. على سبيل المثال، تقنيات مثل خلية الفرز المغناطيسي مع المتاحة تجاريا ممغطس أجهزة ماكينتوش-النانوية سهلت تطوير مناهج جديدة للبحوث والتطبيقات السريرية 22،29. وعلاوة على ذلك، وقد أثبتت الدراسات والأبحاث الحديثة مقارنة الجيل العاصمة من أساليب الالتزام الوحيدات والتكنولوجيا بالاعمال نقاء العاصمة العالي وقابلية استخدام أجهزة ماكينتوش فصل حيدات 22،30. ويعرض الدراسة الحاليةمقارنة بين طريقتين لتوليد البلدان النامية الإنسان من وحيدات معزولة عن PBMCs: 1) عزل الوحيدات من الالتزام و2) عزل الوحيدات عن طريق الانتقاء السلبية باستخدام طقم تخصيب الوحيدات الإنسان التجارية. وتقدم هذه الدراسة أدلة تثبت أن اختيار الإجراء والفصل المغناطيسي السلبي لعزل حيدات يولد أعلى عائد من وحيدات مع عدم وجود اختلافات كبيرة في جدوى الوحيدات بالمقارنة مع حيدات معزولة بسبب طريقة الالتزام. في المقابل، وبعد سبعة أيام، وحيدات معزولة بواسطة مغناطيس متباينة في MDDCs مع قدرة التكاثري أعلى بكثير وأعلى كمية من الخلايا معربا عن إيجابية مزدوج (CD11c و+ / CD14 +) النمط الظاهري دون التأثير على قابلية MDDC. وعموما، تختلف الدراسة الحالية من الدراسات السابقة المشار إليها أعلاه لأنه يدل على قدرة كل من تقنيات لتوليد في وقت واحد وحيدات القادرة على التكاثر والتفريق في CD11c و+MDDCs (&gt; 70٪) بعد سبعة أيام في الثقافة دون المساس حيويتها. وبالإضافة إلى ذلك، يوفر النهج الحالي لتوصيف أول مرة من CD11c ومختلف / السكان CD14 MDDCs عن طريق التدفق الخلوي التصوير. وخلاصة القول، منذ البلدان النامية تلعب دور أساسي فيما يتعلق البحوث في مجال علم المناعة، يجب أن تؤخذ العوامل المختلفة في الاعتبار عند النظر في كيفية تشتق منها وما هي الأساليب المستخدمة لعزل والثقافة لهم في المختبر. ولذلك، تهدف هذه الدراسة إلى تقديم رؤى على طريقتين مختلفتين من العزلة الوحيدات وكيف أن هذه الأساليب تؤثر بشكل مختلف الجدوى الوحيدات والعائد تؤثر في نهاية المطاف شجيري بقاء الخلية، والانتشار، والنمط الظاهري. وسوف تسهم هذه النتائج إلى حد كبير في مجال علم المناعة، وسوف توفر بروتوكول مفصلة من العاصمة العزلة، وتنقية، والتوصيف.

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			<td height="34" style="height:34px;width:535px;">Ficoll-Paque</td>
			<td style="width:228px;">GE Healthcare</td>
			<td style="width:231px;">17-5442-03</td>
			<td style="width:443px;">Must be used at room temperature</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Phosphate-buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">ACK Lysing buffer</td>
			<td>Quality Biological</td>
			<td>118-156-101</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">RPMI 1640 medium</td>
			<td>Life Technologies</td>
			<td>22400-089</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Antibiotic-Antimycotic (100X)</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">RoboSep buffer</td>
			<td>StemCell</td>
			<td>20104</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">EasySep Human monocyte enrichment kit</td>
			<td>StemCell</td>
			<td>19059</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">TC20 Automated cell counter</td>
			<td>Bio-Rad</td>
			<td>145-0101</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">FITC-Dextran</td>
			<td>Sigma Aldrich</td>
			<td>FD4-100MG</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Trypan blue stain (0.4%)</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
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		<tr height="34">
			<td height="34" style="height:34px;">Synergy 2 multi-mode reader</td>
			<td>Biotek</td>
			<td>7131000</td>
			<td></td>
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		<tr height="34">
			<td height="34" style="height:34px;">XTT Sodium salt bioreagent (XTT)</td>
			<td>Sigma Aldrich</td>
			<td>X4626-100MG</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Dimethyl sulfoxide bioreagent (DMS)</td>
			<td>Sigma Aldrich</td>
			<td>D8418-500ML</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Thiazolyl blue tetrazolium bromide (MTT)</td>
			<td>Sigma Aldrich</td>
			<td>m5655-500MG</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td>Bio-Rad</td>
			<td>161-0302</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Phenazine Methosulfate (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>P9626-1G</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Inactivated (HI) Human Serum</td>
			<td>Chemicon</td>
			<td>S1-100ML</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Accuri C6 Flow Cytometer</td>
			<td>BD Accuri</td>
			<td>653119</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">FlowSight Amnis Flow Cytometer</td>
			<td>EMD Millipore</td>
			<td>100300</td>
			<td></td>
		</tr>
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characterization of human monocyte-derived dendritic cells by imaging flow cytometry: a comparison between two monocyte isolation protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

<html> الوحيدات العائد من الفصل المغناطيسي والعالي بالمقارنة مع الوحيدات الغلة عن طريق أسلوب الالتزام البيانات الواردة في الشكل 1 عرض PBMC وعدد خلايا الوحيدات من خلال طريقة الاست...

Watch this Scientific Journal Video about Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols at JoVE.com

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

توصيف الخلايا الجذعية البشرية الوحيدات المستمدة من التصوير التدفق الخلوي: مقارنة بين اثنين من بروتوكولات الوحيدات عزل

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

The overall goal of this study is to compare two different methods of human monocyte isolation for dendritic cell generation in vitro and to characterize the CT11c, CT14 cell surface expression of the resulting monocyte derived populations by imaging flow cytometry. These monocytes isolation protocols can contribute to the development of reliable methods for the purification of human dendritic cells for research and clinical applications. The main advantage of these protocols is that they facilitate the isolation of human monocytes and their differentiation into viable and functional monocyte derived cells.In addition, this is the first study to characterize the specific population of monocyte derived dendritic cells by single cell imaging flow cytometry. This method can also be applied as an alternative for the isolation of other rare cells at an adequate yield for downstream research applications. Visual demonstration of these methods is critical as it can be challenging to handle the blood samples safely without the appropriate instruction and experience.Begin by diluting the blood sample to a one to one ratio with PBS in a T75 flask. Then, add 15 milliliters of density gradient solution into a 50 milliliter centrifuge tube and carefully layer 25 to 30 milliliters of the diluted blood over the gradient. To achieve a proper separation of the peripheral blood mononuclear cells, load the blood onto the density gradient solution quickly, but carefully and without mixing the layers.Separate the cells by centrifugation, followed by the transfer of the white blood cell interface layer into a new 50 milliliter conical tube. Wash the cells two times in PBS, re-suspending the final pellet in 10 milliliters of ammonium chloride potassium lysing buffer for 15 minutes at 4 degrees celsius. Then wash the cells two more times in PBS.To isolate monocytes by the adherence method, culture five times 10 to the seven cells of the resulting PBMC pellet per 10 milliliters of complete medium in a new T75 flask for two hours at 37 degrees celsius and five percent carbon dioxide in a humidified incubator. At the end of the incubation, discard the non-adherent floating cells and gently wash the adherent cells two times with PBS. Then, feed the adherent cells with complete cell culture medium supplemented with human GMCSF and IL4 and return the culture to the incubator for five to seven days.To isolate the monocytes by magnetic separation, after collecting the PBMC by density gradient separation transfer the white blood cell layer to a five milliliter polystyrene tube and re-suspend the cells in PBS at a five times 10 to seven cells per milliliter concentration. Next, add 50 microliters of a human monocyte enrichment cocktail per milliliter to the cells with mixing and incubate the cells with the cocktail at four degrees celsius for 10 minutes. Then add 50 microliters of magnetic particles per milliliter of cells with careful mixing and incubate the cells at 4 degrees celsius for five minutes.At the end of the second incubation, add PBS to the cells to bring the total volume up to two point five milliliters and mix by pipetting two to three times. Then place the polystyrene tube in an appropriate magnetic device at room temperature. After two and a half minutes, with the tube still in the magnet, decant the purified monocyte fraction into a new 15 milliliter conical tube.Then culture the purified monocytes and complete cell culture medium supplemented with GMCSF and IL4 for five to seven days as just demonstrated. After seven days of differentiation, dispense one times 10 to the six cell aliquats of the monocyte derived dendritic cells into the appropriate number of one point five milliliter tubes, and add 50 microliters of heat inactivated human serum to each sample to block any non-specific finding. After 10 minutes, centrifuge the cells and re-suspend the pellets in the appropriate fluorescence conjugated primary antibodies for 20 minutes at four degrees celsius protected from light.Then wash the cells two times in one milliliter of PBS, resuspending the pellets in 100 microliters of PBS per one times ten to the six cells. Just before the analysis, add one microliter of DAPI to each tube. Then, read the samples on a single cell imaging flow cytometer according to the manufacturer's instructions.On average, monocytes isolated by the adherence method account for approximately six point two percent of the total peripheral blood monocyte cell, or PBMC, population, while the monocytes isolated by magnetic separation account for up to 25 percent of the total PBMC. Monocytes isolated by either method are equally viable. MDDCs differentiated from monocytes purified by both methods were first gated based on DAPI negative or viable cells from total single cells, followed by gating of each cell population.Both isolation methods yielded a low single CD14 positive population and both methods yielded a high total CD11c positive population. Further phenotypic analysis was performed on all the CD11c positive cells and even though magnetic isolation gives a higher percentage of double positive CD11c positive CD14 positive cells, this effect was non-significant when compared with the adherence method. These double positive CD11c positive, CD14 positive cells may be early stage differentiated MDDCs that have not yet dropped the CD14 monocytic marker.When analyzing the single CD11c positive cells, the adherence method gives the highest yield of CD11c positive, CD14 negative cells. These single positive cells may be late stage differentiated MDDCs. Once mastered, the adherence and magnetic isolation techniques can be completed in three to four and one to two hours respectively if they are performed properly.While attempting this procedure, it's important to remember to have fresh dendritic cell differentiation cytokines every 48 hours when the medium is replenished. Working with biohazard fluids such as blood, can be extremely harmful, and the use of appropriate personal protective equipment and disposal methods should always be taken when performing these procedures. This study paves the way for researchers in the field of immunology to explore the use of human monocytes and dendritic cells for therapeutic treatment.After watching this video, you should have a good understanding of how to optimize human monocyte isolation for generating different populations of monocyte derived cells in vitro.

Research

JoVE Journal

Immunology and Infection

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

تفعيل وقياس NLRP3 Inflammasome آخر عن طريق IL-1β في الخلايا الجذعية المشتقة من الوحيدات الإنسان

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

جيل من الوحيدات المستمدة الخلايا الجذعية البشرية من الدم الكامل

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

تحليل الخلايا الدبقية الصغيرة والبلاعم المستمدة من الخلايا العصبية من الجهاز العصبي المركزي عن طريق التدفق الخلوي

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

العزلة والثقافة في المختبر من البلاعم السنخية مورين والبشرية

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

وصف مجموعات فرعية الوحيدات البشرية بالدم كله يتدفق التحليل الخلوي

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

تحديد كمية النشاط الكيميائي للخلايا الأحادية في الجسم الحي وتوصيف الضامة المشتقة من خلايا الدم

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

سيرنا اليكتروبوريشن لتعدل اوتوهاغي في الهربس البسيط الفيروسات نوع 1-الخلايا الجذعية المصابة بالبويضات المشتقة

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas ...

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection

استقطاب M1 و M2 الإنسان الخلايا المشتقة من Monocyte وتحليل مع تدفق قياس الخلايا على عدوى السل المتفطرة

Human macrophages are primary host cells of intracellular Mycobacterium tuberculosis (Mtb) infection and thus have a central role in immune control of ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

تصور الكاسباز الالتهابي الناجم عن القرب في البلاعم المشتقة من الخلايا الأحادية البشرية

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

تحديد مناعة اللقاح باستخدام الخلايا المتغصنة المشتقة من وحيدات الأبقار

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...