Bu proje, Ulusal Sağlık Grants HL087088 (MK) Enstitüleri ve HL18208 (MK) tarafından desteklenmiştir.

1. İnsan T Lenfositler izolasyonu <ol><li> Insan sağlıklı bir donörden kan alın. Kan, bir sonraki adıma geçmeden önce oda sıcaklığında (~ 30 dk) soğumasını bekleyin. </li><li> 8 ml yuvarlak alt polistiren tüp yavaşça içine oda sıcaklığında polimorf yoğunluk gradiyenti medya 3 mL pipetle. Polimorf medya üstüne 3 ml tam kan yavaşça ekleyin. Iki reaktifler karışmasını önlemek için önemlidir. </li><li> Tüpler oda sıcaklığında 45 dakika boyunca 500 xg&#39;de santrifüjleyin. </li><li> Santrifüj sonrasında, periferik kan mononükleer hücreleri (hücre) üst hücre katmanı içine diğer kan bileşenleri ayırdığım. Hücre katmanı, ilk bulutlu grup olarak, yukarıdan aşağıya doğru görünür. </li><li> PBMC katmanının üstüne kan açık sarı renkli üst faz, dikkatlice çıkarın ve sonra bir 15 ml veya 50 ml konik tüp PBMC tabaka transfer etmek için P1000 mikropipet kullanın. </li><li> 5 dakika her zaman için 500 xg&#39;de hücreler santrifüj, PBMC PBS ile iki kez yıkayın. Süpernatant her yıkamadan sonra biraz bulutlu olacak. </li></ol> 2. İnsan T Lenfositler Kültür <ol><li> Bir pipet kullanarak,% 10 FBS içeren 20 ml RPMI 1640 medya T-75 kültür şişelerinde PBMC transferi,% 1 penisilin / streptomisin ve 1 mcg / ml fitohemaglütininle (PHA). </li><li> 37 ° ° C&#39;de ve% 5 CO 2 için en az 1 saat ve 24 saate kadar. Bu adım, monositler, süspansiyon kalır lenfositler ayrılmış, balon yüzeye yapışık olacak sağlar. Bu aşamada kısa bir inkübasyon (1 saat) kullanılması durumunda, adım 2.1 &#39;de belirtilen PHA ile ilave% 10 FBS ve% 1 penisilin / streptomisin içeren RPMI 1640 medya kullanımı için kabul edilebilir. </li><li> Dikkatle şişesi tüm medya kaldırmak 50 ml konik tüp ekleyin ve 5 dakika boyunca 500 xg&#39;de santrifüj. </li><li> Şimdi öncelikle lenfositler içeren hücre pelet, süspanse edin ve% 10 FBS içeren 25 ml RPMI 1640 medya içeren yeni bir T-75 balon hücreleri transferi,% 1 penisilin / streptomisin ve 1 mcg / ml PHA. </li><li> 3 gün (2 gün hücre ilk kuluçka gecede ise) 37 ° C&#39;de inkübe edin. 24 saat sonra büyüme, 15-20 ml taze medya ve daha büyük bir T-175 şişesi transfer için gerekli olabilir. </li><li> 3 gün sonra, 50 ml konik tüp şişesi ve transfer askıya medya ve lenfositler kaldırmak için bir pipet kullanın. 5 dakika boyunca 500 xg&#39;de santrifüjleyin. </li><li> % 10 FBS ile 25 ml (T-75) veya 50 ml RPMI 1640 (T-175) içeren yeni bir T-75 veya T-175 şişesi hücre pelet ve transfer hücreleri tekrar süspansiyon hale getirin,% 1 penisilin / streptomisin ve 20 ng / ml insan IL-2 veya IL-15. </li><li> 4-7 gün lenfositler büyütün. Eğer bir T-75 şişesi ile başlayan, kültür, 1-2 gün sonra genişletilmiş ve bir T-175 balona transfer edilmesi gerekecektir. </li></ol> 3 İn Vitro Lenfosit Göç Testi <ol><li> 1 gün önce göç tahlil, mont, 4, 20 mg / ml Protein PBS içinde A veya G gecede bir cam alt 0.17 mm çanak ° C </li><li> PBS ile yoğun bulaşık yıkayın. </li><li> Insan ICAM-1/Fc (10 mg / ml) ve insan çanak PBS çözüm bu reaktifler ekleyerek ve 4 saat oda sıcaklığında inkübe SDF-1 (2 mcg / ml). Hareketsiz </li><li> T lenfositleri, ilk T-175 şişesi hemasitometre kullanarak kültür hücre yoğunluğu tespit ederek hazırlayın. Yaklaşık 2-5 x 10 5 hücreleri göç analizi için uygun bir hücre yoğunluğu elde etmek için çanak için kullanılmalıdır. </li><li> T lenfositlerin iki kez PBS ile yıkayın ve sonra, 1 mg / mL D-glukoz içeren 1 ml L-15 medya tekrar süspansiyon haline getirin. </li><li> Yoğun PBS ile ICAM-1/Fc ve SDF-1 ile kaplı çanak yıkayın. </li><li> 1 ml medya çanak T lenfositleri aktarın ve 37 ° onları korumak ° C </li></ol> 4. NIS Elements Yazılım Kullanarak Bir Resim Sırası yakalama <ol><li> NIS Elements yazılımı açın. </li><li> Kamera ayarları menüsünde, hem de canlı görüntüleme ve görüntü yakalama modu olarak &quot;2x2 binning&quot; seçin. </li><li> Uygulamalar menüsüne gidin ve Çalıştır / tanımlayın Deney seçin. </li><li> Görüntü ve görüntü yakalama sırası toplam uzunluğu arasındaki zaman uzunluğu seçin. </li><li> Görüntü elde başlamak için &quot;Çalıştır&quot; düğmesine basın. </li><li> Hücreler takip edilebilir ve göç parametreleri (hız, yol uzunluğu, yer değiştirme, vb.) ImageJ, AutoQUANT veya Volocity (Şekil 1) de dahil olmak üzere çeşitli yazılım paketleri, birini kullanarak nicel. </li><li> Oluşturmak için &quot;örümcek web arsa, her hücre ve timepoint toplanır (Şekil 2) ve kökeni her hücre için ortak bir başlangıç ​​noktası (Şekil 3) ile bir grafik yansıtılıyor.&quot; Xy koordinatları. </li></ol> 5. Temsilcisi Sonuçlar IL-2 veya IL-15, T lenfositleri ya varlığı kültür, 6 günyanı sıra olumlu CD4 ve / veya CD8 boyama pozitif CD3 boyama tarafından belirlenir hücrelerinin&gt;% 98 kadar olun. IL-2 için, biz% 83 CD4 + hücreler,% 15 CD8 + ve &lt;% 1 CD4 + CD8 + hücreler bulundu. IL-15 için,% 88 CD4 + hücreler, CD8 +% 11 ve &lt;% 1 CD4 + CD8 + hücreler bulundu. ICAM-1/SDF-1 yüzeylerde T lenfosit göç sırasında, hücreler, yaklaşık 1 saatlik zaman dilimi içinde sürekli 15 mm / dak bir hız sergiledi. ICAM-1/SDF-1 T lenfosit göç, LFA-1-aracılı adezyon bağımlı olarak bir anti-LFA-1 ligand engelleme antikor göç ciddi engeller. <img src="/files/ftp_upload/2017/2017fig1.jpg" alt="Şekil 1" /> Şekil 1. T lenfositleri göç Hücre izleme, tam kan izole edilen ve 6 gün boyunca IL-2 veya IL-15 varlığında kültür T lenfositleri 10 mcg / mL ICAM-1 ve kaplamalı cam tabanlı yemekler yapışmaz ve geçiş için izin verildi 2 mcg / ml SDF-1. Görüntüler 30 dakika boyunca her 10 saniyede elde edildi. Hücreler her görüntünün Volocity yazılım kullanarak zaman içinde tespit ve takip edilmiştir. Bu film ~ 15 mm / dak hızda T lenfositlerin rastgele göç gösterir. <a href="http://www.jove.com/files/ftp_upload/2017/2017_Kim_Movie1.mov">Videoyu görmek için buraya tıklayın.</a> T lenfositleri göç Film 1. Hücre izleme. T lenfositlerin, tam kan varlığında izole ve kültür IL-2, 6 gün süreyle veya IL-15, 10 mikrogram / ml ICAM-1 ve 2 mcg / ml SDF-1 ile kaplı cam dipli yemekleri üzerine yapışır ve göç etmeye izin verildi . Görüntüler 30 dakika boyunca her 10 saniyede elde edildi. Hücreler her görüntünün Volocity yazılım kullanarak zaman içinde tespit ve takip edilmiştir. Bu film ~ 15 mm / dak hızda T lenfositlerin rastgele göç gösterir. <img src="/files/ftp_upload/2017/2017fig2.jpg" alt="Şekil 2" /> Şekil 2 Spatiotemporal hücre konumu. Her bir hücrenin XY koordinatlarını Volocity yazılımı kullanarak her zaman noktası için elde edilmiştir. <img src="/files/ftp_upload/2017/2017fig3.jpg" alt="Şekil 3" /> Şekil 3 &quot;Örümcek web arsa.&quot; XYt kullanarak, her bir hücre, 15 hücre rastgele seçilmiştir ve kökeni ortak bir başlangıç ​​noktası ile çizilen koordine eder. Örümcek web grafikleri karşılaştırarak, farklı deneysel koşullar arasında göç farklılıkları hızlı bir görsel tasviri verir. T lenfosit göçü kontrol koşulları altında (sol panel) ve anti-LFA-1 ligand engelleme antikor (sağ panel) varlığı Arsalar.

<ol>
	<li>Hyun, Y. M., Chung, H. L., McGrath, J. L., Waugh, R. E., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+integrin+VLA-4+localizes+to+the+lamellipodia+and+mediates+t+cell+migration+on+VCAM-1.">Activated integrin VLA-4 localizes to the lamellipodia and mediates t cell migration on VCAM-1.</a> J Immunol. 183, 359-369 (2009).</li><li>Morin, N. A., Oakes, P. W., Hyun, Y. M., Lee, D., Chin, Y. E., King, M. R., Springer, T. A., Shimaoka, M., Tang, J. X., Reichner, J. S., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonmuscle+myosin+heavy+chain+IIA+mediates+integrin+LFA-1+de-adhesion+during+T+lymphocyte+migration.">Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration.</a> The Journal of experimental medicine. 205, 195-205 (2008).</li><li>Hyun, Y. M., Lefort, C. T., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+integrins+and+their+ligand+interactions.">Leukocyte integrins and their ligand interactions.</a> Immunologic research. , (2009).</li></ol>

Bu deneyde, birincil insan T lenfosit motilite analiz etmek için basit bir sistem hakkında ayrıntılı bilgi sağlar. In vitro göç deneyleri, çeşitli hücre tipleri hareketine katılan birçok moleküller ve sinyal yolları rolleri incelemek kullanılmıştır . Bizim protokol kendi deney tasarımı akılda tutulması gereken bazı kritik kontrolleri şunlardır: 1), sığır serum albumini (BSA) veya poli-L lisin (PLL) gibi yapışkanlı olmayan veya non-integrin yapışkan substrat kaplamalar, sırası...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11875</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH300070.03</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>1-Step Polymorphs</td>
<td>&nbsp;</td>
<td>Accurate Chemical</td>
<td>AN221725</td>
<td>&nbsp;</td>
<tr>
<td>PHA</td>
<td>&nbsp;</td>
<td>Remel/Fisher Scientific</td>
<td>R30852801</td>
<td>&nbsp;</td>
<tr>
<td>Rec. human IL-2</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>202-IL</td>
<td>&nbsp;</td>
<tr>
<td>Rec. human IL-15</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>247-IL</td>
<td>&nbsp;</td>
<tr>
<td>Protein G</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>19459</td>
<td>&nbsp;</td>
<tr>
<td>Rec. human ICAM-1/Fc</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>720-IC</td>
<td>&nbsp;</td>
<tr>
<td>Rec. human SDF-1</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>350-NS</td>
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human t lymphocyte isolation, culture and analysis of migration in vitro

T lenfositlerin göçü, diğer hücrelere veya ekstraselüler matriks proteinleri ile ifade ligandlar hücre yüzeyinde integrinler yapışkan bir etkileşim içerir. Düşük bir yakınlık durumuna hücre öncü afinitesi yüksek bir devlet İntegrinlerin hassas uzaysal aktivasyonu, T lenfosit geçiş için önemli<sup&gt; 1</sup&gt;. Aynı şekilde, hücre firar kenarına çekilmesi, ya da uropod, kalıcı integrin-bağımlı T lenfosit motilite korunmasında gerekli bir adım.<sup&gt; 2</sup&gt;. Lökositlerin aşırı istihdam ve göç inhibe etmek için bir araç olarak otoimmun veya inflamatuar hastalıklar hedef integrinler Birçok terapötik yaklaşımlar<sup&gt; 3</sup&gt;. Insan T lenfosit göçü düzenleyen moleküler olayları incelemek için, biz bir şekilde kullanmış olması<em&gt; In vitro</em&gt; T lenfosit damar alımı sırasında karşılaştığı ortamı taklit eden bir iki boyutlu bir substrat hücre göçü analiz sistemi. T lenfositleri ilk insan donörlerden izole edilir ve yedi ile on gün sonra uyarılmış ve kültür. Tahlil sırasında, T lenfositleri hücrelerarası adezyon molekülü-1 (ICAM-1), LFA-1 integrin için bir ligand ve stromal hücre kaynaklı faktör-1 (SDF-1) ile kaplı bir substrat üzerine yapışır ve göç etmeye izin verilir. Bizim verilerimiz, T lenfositleri ~ 15 mm / dak bir göç hızı sergileyen olduğunu göstermektedir. T lenfosit göç integrin abluka tarafından inhibe olabilir<sup&gt; 1</sup&gt; Veya hücresel actomyosin makine inhibitörleri ile hücre göçü düzenleyen<sup&gt; 2</sup&gt;.

Watch this Scientific Journal Video about Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro at JoVE.com

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lenfosit göç homing lenfoid organlar, damar çıkış sırasında oluşur ve periferik dokularda girmeden. Burada, T lenfosit göçü analiz etmek için kullanılan bir protokol tarif<em&gt; In vitro</em&gt;.

Göç İnsan T Lenfosit İzolasyon, Kültür ve Analiz In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

human-t-lymphocyte-isolation-culture-analysis-migration

Research

JoVE Journal

Immunology and Infection

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

46.1K Görüntüleme.  University of Rochester. T lenfosit göç homing lenfoid organlar, damar çıkış sırasında oluşur ve periferik dokularda girmeden. Burada, T lenfosit göçü analiz etmek için kullanılan bir protokol tarif In vitro.

Video: Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

Primer insan T hücrelerinin piggyBac Transposon Sistem Modifikasyon

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

İmmünoloji ve Enfeksiyon

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Fenotipik Analizi ve İnsan Cilt lenfosit izolasyonu<em&gt; Ex Vivo</em&gt; Hücre Kültürü

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Fare Naif CD4<sup&gt; +</sup&gt; T Hücre İzolasyonu ve<em&gt; In vitro</em&gt; T hücre alt içine Farklılaşma

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

Iltihaplı Dermisinde içinde görüntüleme CD4 + T hücre Geçiş Göç

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

İnsan Tüm Kan Natural Killer Hücre Parçalayıcı Faaliyet Akışı Sitometrik Analizi

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

İnsan Endoservikal Gamma Delta T Hücreleri İzolasyon ve Akım Sitometrisi Analizi

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Bir kullanma Lenfosit Ekstravazasyonunun analizi<em&gt; İn Vitro</em&gt; İnsan Kan-beyin bariyeri Model

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

İnsan monosit kemotaksisi Vitro ve In Vivo kaçakçılığı fare lenfosit miktarının

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Yalıtım ve fare ve insan alveoler makrofajlar Vitro kültürü

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

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Yamultma akış kaynaklı geçiş antikorundan marjinal bölge B hücrelerinin in Vitro analizine

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...