Bu yazıda, Ulusal Sağlık ve Tıbbi Araştırma Konseyi (NHMRC), Avustralya Programı Hibe (CRP) ve NHMRC Projesi Hibe (CRP ve BJCQ) tarafından desteklenmiştir. Ayrıca BJCQ NHMRC Peter Doherty doktora sonrası çalışmalarını yürütüyor.

1) Reaktif Kurulum 1.1) KAKE stok solüsyonu KAKE boya genellikle 25 mg flakon carboxyfluorescein diasetat succinimidyl ester (CFDA, SE) (MW 557,47 g / mol) olarak satın alınmaktadır. (-20 ° C&#39;de birkaç ay için 50-100 mcL alikotları olarak saklamak) 5 mM nihai bir stok solüsyonu için 8.96 mL DMSO 25 mg çözülür. Depolama sırasında bu tür bir poz kaçınılmalıdır kritik böylece CFDA, SE, sulu çözelti ile reaksiyona girer. 1.2) Lenfositler Farelerin dalak ve lenf düğümleri ile 1 ml kültür ortamı (örneğin RPMI) lenfositlerin yalıtın ve tekrar süspansiyon arasında 0.5 x 10 6 hücre konsantrasyonu ile fetal buzağı serumu (FCS), - 10% 5 ısı ile inaktive edilmiş (HI) x 10 7 / ml. 2) KAKE Etiketleme 2.1) Rutin yöntemi: <ol class="lalpha"><li> Dikkatlice taze (ıslatılmış) 10 ml konik tüp alt, orta ve yerin 1 ml hacmi iyice hücreleri tekrar süspansiyon haline getirin. </li><li> (Islak olmayan bir tüp kullanarak hareketli ve erken CFDA, GD çözümler ile karıştırılarak 1 ml hücre süspansiyonu önleyecektir) tüp yatay yatırın. </li><li> Hücre çözümü ile temas yapmaz sağlanması tüpün üstündeki plastik olmayan ıslak kısmını dikkatlice PBS 110 mcL ekleyin. </li><li> 110 mcL PBS CFDA, GD, 5 mM stokunun 1.1 mcL tekrar </li><li> Hızla tüp kapağı ve çözümleri hızlı üniforma karıştırma almak için de ters ve vorteks. KAKE boya 5 mcM son bir konsantrasyonda olduğunu unutmayın, ancak, optimum konsantrasyonda hazırlanan her parti için belirlenecek, ve ardından etkili olduğundan emin olmak için her 6 ayda bir kontrol. </li><li> Hücrelerin oda sıcaklığında 5 dakika inkübe edin. CFDA, SE KAKE dönüştürülmesi üzerine (tartışma) boya, floresan ve böylece aşırı ışığa maruz tarafından beyazlatma eğilimli hale gelir. Bu nedenle, örneğin, alüminyum folyo ile kaplayıp, bu noktadan itibaren tüp ışıktan korumak için tavsiye edilir. </li><li> Hücreleri, 20 ° C ve süpernatant atarak 5 dakika boyunca 300 xg&#39;de santrifüj yoluyla sedimenting, 20 ° C PBS% 5 HI FCS içeren 10 ciltlik seyreltilmesi ile yıkayın. Iki kez daha yıkayın tekrarlayın. </li></ol> (10 daha yüksek hücre sayıları için de uygun olan 2.2) Alternatif yöntem, 30 x 10 7) : <ol class="lalpha"><li> 5 mM stok 2 mcL 1 PBS ml ve 1 ml tamamen yeniden süspanse hücrelerin hızlı bir şekilde bu eklentiyi ekleyerek 2 x CFDA (10 mcM), GD çözüm hazırlayın, sonra hızlı bir şekilde tüpün kapağı ve ters ve vorteks almak hızlı üniforma karıştırma. </li><li> Hücrelerin oda sıcaklığında 5 dakika inkübe edin ve adımları 2.1 (f) ve 2.1 (g) olarak yıkayın. </li></ol> 2.3) Hücreler daha sonra hücre bölünmesi indüksiyonu için bir ilgi protokolü uygulanabilir. Etiketli hücreleri hem de in vitro ve in vivo testleri kullanılabilir. 2.4) yayılması testin bitiminde, hücre hasat ve flow sitometri ile analiz (temsili örnekler için aşağıya bakınız). 3) Temsilci Sonuçlar KAKE seyreltme lenfosit proliferasyonunu değerlendirmek için, bölünmemiş hücrelerinin (yani maksimum floresans) ve olmayan etiketli hücreleri (yani en az floresan) floresan bilmek yararlıdır. Bu nedenle, deney tasarımı, bu kontroller dikkate almalıdır. Aşağıda, in vitro ve in vivo deneylerde flow sitometri CD8 + T hücre bölünmesi ölçmek için KAKE kullanarak örnekler. 3.1 in vitro stimülasyon) Şekil 1 KAKE profilleri gösterir KAKE-etiketli ovalbumin (OVA)-spesifik T hücre reseptörü transgenik OVA çeşitli miktarlarda darbeli dendritik hücreler ile 3 gün kültür sonra CD8 + OT-T hücreleri. CD8 + T hücreleri OT-I farelerin dalak ve lenf düğümleri arınmış KAKE ve 3.3 x 10 4 DC kültürlü 1 x 10 5 hücreleri ile etiketlenir. DC 37 ° C 1 saat için OVA ile darbeli ve kültüre önce iki kez yıkanır. 3 gün sonra, hasat ve etiketli anti-CD8-APC antikorlar ve Hoechst 33.258 ve daha sonra analiz flow sitometri (50.000 olaylar toplanan) hücreler. Live (Hoechst 33.258 -) CD8 + hücreler gösterilmiştir. Etiketli hücre hücre ve sınırlarını tespit hücre bölünmeleri floresan otomatik gösterirken, kontrol grubu olarak, tek başına kültür CD8 + T hücreleri, olmayan bölünen hücrelerden KAKE yoğunluğunu gösterir. 4 KAKE doruklarına, bu örnekte, paneller her hücreleri 3 bölümler uğramadığını belirten görülebilir olduğunu unutmayın. 3.2 in vivo stimülasyon) Şekil 2 transgenik CD8 + OT-T hücrelerinin in vivo olarak 3 gün sonra KAKE etiketli OVA-spesifik T hücre reseptör KAKE profilleri gösterir In 20 mg OVA varlığı. CD8 + T hücreleri CD45.1 congenic OT-I farelerin dalak ve lenf düğümleri arınmış KAKE ve 5 x 10 6 hücre içine iv enjekte C56BL/6J lateral kuyruk ven (CD45.2 +), farelerde ile etiketlenir . , 2 saat fareler sonra 20 mg OVA iv enjekte edildi. 3 gün sonra fareler dalak, anti-B220-PerCP anti-CD8-APC-Cy7 ve anti-CD45.1-PE-Cy7 antikorlar ve Hoechst 33.258 ile etiketlenmiş, bir tek hücre süspansiyonu haline getirilmiş, toplanan ve daha sonra analiz edildi flow sitometri (1.000.000 olaylar toplanan). Live (Hoechst 33.258 -) B220-hücreleri sağ panelde gösterilen sol paneli ve kapılı CD8 + CD45.1 + hücreler gösterilmiştir. Kontrol grubu olarak, unstimulated CD8 KAKE etiketli + T hücreleri, bölünen hücrelerden KAKE yoğunluğunu gösterir olmayan etiketli hücreleri, hücreler ve sınırlarını tespit hücre bölünmeleri otomatik floresan gösterir. CD45 allotypic fark devredilen konak çözme CD8 + T hücreleri, toplam CD8 + T hücreleri (sol panel) küçük bir alt kümesi olarak hayati olduğunu unutmayın. En hücreler hücreleri 6 bölünmelere uğramadığını belirterek, bu örnek (sağ panel) 7 KAKE doruklarına giren unutmayın. <img src="/files/ftp_upload/2259/2259fig1.jpg" alt="Şekil 1" /> Şekil 1. KAKE-işaretli ovalbumin (OVA)-spesifik T hücre reseptör transgenik CD8 Not OVA, farklı konsantrasyonları ile darbeli DC in vitro cevap için + OT-I T hücreleri,, 3 gün kültür sonra gösterilen veriler. CD8 olmayan-bölünmüş nüfus + T yeteneği antijen (açık gri histogram) ve oto-floresan etiketli olmayan nüfus (koyu gri histogram) yokluğunda hücre. Şekil gösteriyor CD8 + T hücreleri, T hücreleri yüksek antijen konsantrasyonlarda bölünmesi, KAKE seyreltme doruklarına göre 1-3 kez bölünmüş var. <img src="/files/ftp_upload/2259/2259fig2.jpg" alt="Şekil 2" /> Şekil 2. Yeteneği KAKE-işaretli ovalbumin (OVA)-özel adoptively OVA için cevap C57BL / 6 farelerinde içine transfer T hücre reseptör transgenik CD8 + OT-I T hücreleri,, evlatlık transferi sonrası 3 gün gösterilen veri Sol paneli:. CD8 +, CD45. alıcı farelerde 1 + OT-1 T hücreleri. Sağ panel: KAKE çoğalması profili. Not CD8 olmayan bölünmüş nüfus + alıcı farelerde T hücreleri antijen (açık gri histogram) ve otomatik olmayan etiketli lenfositleri (koyu gri histogram) floresan almıyor. Şekil KAKE seyreltme doruklarına göre 1-6 kez bölünmüş CD8 + T hücreleri gösteriyor. <img src="/files/ftp_upload/2259/2259fig3.jpg" alt="Şekil 3" /> Şekil 3. Tartışma metin carboxyfluorescein diasetat succinimidyl ester (CFDA, SE) ile hücrelerin etiketleme sırasında meydana gelen çeşitli moleküler olayların bir temsili. Süreçle ilgili ayrıntılar için bkz. Not: genel kısaltma &#39;KAKE&#39; değil, CFDA, SE, genel etiketleme reaktif ve etiketleme işlemi tanımlamak için kullanılır. Parish 4 dayalı Şekil.

<ol><li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determination+of+lymphocyte+division+by+flow+cytometry%5BTitle%5D)+AND+%22J.+Immunol.+Methods%22%5BJournal%5D)">Determination of lymphocyte division by flow cytometry.</a> J. Immunol. Methods. 171, 131-137 (1994).</li>
<li>Parish, C. R., Glidden, M. H., Quah, B. J., Warren, H. S. Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aParish%2C+CR+%22Current+protocols+in+immunology%22">Current protocols in immunology</a>. Coligan, J. E. , (2009).</li>
<li>Quah, B. J., Warren, H. S., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.</a> Nature Protocols. 2, 2049-2056 (2007).</li>
<li>Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescent+dyes+for+lymphocyte+migration+and+proliferation+studies%5BTitle%5D)+AND+%22Immunol+Cell+Biol%22%5BJournal%5D)">Fluorescent dyes for lymphocyte migration and proliferation studies.</a> Immunol Cell Biol. 77, 499-508 (1999).</li>
</ol>

KAKE çalıştığını ve neden hızlı üniforma etiketleme çoğalması analizinde kullanılmak üzere gerekli nasıl takdir için, KAKE hücre etiketleme moleküler temelini anlamak için yararlıdır. Bu iki faz, 1) Hücre giriş ve 2) Protein etiketleme (Şekil 3) ayrılabilir. 1) Hücre giriş: Giriş hücre boya boya diacetylated formu, carboxyfluorescein diasetat succinimidyl ester (CFDA, SE) aracılığı ile. Asetatlar plazma zarından hızlı akı sağlayan boya yüksek membran permeant olun. Böylece hücre içinde bulunan esterazlar, böylece tutunmaya CFDA, SE asetatlar ve daha az membran permeant boya KAKE formu doğuran, hücre içinde konsantre boya. 2) Protein etiketleme: CFDA, SE ve KAKE hem de amino-reaktif succinimidyl yan zincirler olmasına rağmen, sadece KAKE floresan. KAKE yüksek hücre içi konsantrasyonu hızlı ve proteinlerin yüksek düzeyde hücre içi etiketleme kolaylaştırır. Hücre KAKE etiketleme temsilcisi sonuçları (Şekil 1 ve 2) &#39;de gösterildiği gibi birçok bölünmeler geçiren hücreler çözmek için kritik hücre etiketli homojen bir nüfus, elde etmek için hızlı bir şekilde yapılmalıdır.

<table><tbody><tr><td>CFDA,SE</td><td>Molecular Probes, Life Technologies</td><td></td><td></td></tr><tr><td>DMSO</td><td>Cambridge Isotope Laboratories</td><td></td><td></td></tr><tr><td>Lymphocytes</td><td>eg mouse or human</td><td></td><td></td></tr><tr><td>RPMI</td><td>GIBCO, by Life Technologies</td><td></td><td></td></tr><tr><td>FCS</td><td>SAFC Global</td><td></td><td></td></tr><tr><td>10-15ml conical tubes</td><td>Falcon BD</td><td></td><td></td></tr><tr><td>PBS</td><td>GIBCO, by Life Technologies</td><td></td><td></td></tr><tr><td>Aluminium foil</td><td>Capral Aluminium</td><td></td><td></td></tr><tr><td>Vortex</td><td>Scientific Industries Inc.</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Flow cytometer</td><td>BD Biosciences</td><td></td><td></td></tr></tbody></table>

the use of carboxyfluorescein diacetate succinimidyl ester (cfse) to monitor lymphocyte proliferation

Carboxyfluorescein succinimidyl ester (KAKE) lenfosit bölümü 1-3 izlemek için etkili ve popüler bir araç . KAKE kovalent, floresan boya, carboxyfluorescein uzun ömürlü hücre içi moleküller etiketler. Böylece KAKE etiketli hücre bölünürken, onun nesillerinde carboxyfluorescein etiketli moleküllerinin yarısı numarası ile donatılmış ve böylece her bir hücre bölünmesi, Flow sitometri ile hücre floresan karşılık gelen azalma ölçerek tespit edilebilir. Düşük hücre toksisite ile birleştiğinde son derece düşük varyansın yüksek bir floresan yoğunluğu ile lenfosit toplulukları, etiket için KAKE kapasitesi, hücre bölünmesi ölçmek için ideal bir boya olun. Floresein bazlı boya olduğundan, aynı zamanda çok renkli akış sitometri uygulanabilir hale fluorochromes geniş bir yelpazesi ile uyumludur. Bu makale, genellikle fare lenfositler 8 hücre bölünmeleri kadar izleme amaçlı etiketlenmesi için kullanılan prosedürler açıklanmaktadır. Bu etiketli hücreleri in vitro ve in vivo çalışmalar için de kullanılabilir.

Watch this Scientific Journal Video about The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation at JoVE.com

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

KAKE kovalent, floresan boya, carboxyfluorescein uzun ömürlü hücre içi moleküller etiketler. Gibi KAKE etiketli hücre böler, döl ve böylece hücre bölünmesini değerlendirmek için kullanılan floresan yarısı miktarı, var. Bu makalede, tipik olarak KAKE fare lenfositler etiketlenmesi için kullanılan prosedürler açıklanmaktadır.

Lenfosit Silahların Yayılmasının Önlenmesi Monitör Carboxyfluorescein diasetat Succinimidyl Ester (KAKE) Kullanımı

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

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Research

JoVE Journal

Immunology and Infection

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

91.9K Görüntüleme.  John Curtin School of Medical Research, Australian National University. KAKE kovalent, floresan boya, carboxyfluorescein uzun ömürlü hücre içi moleküller etiketler. Gibi KAKE etiketli hücre böler, döl ve böylece hücre bölünmesini değerlendirmek için kullanılan floresan yarısı miktarı, var. Bu makalede, tipik olarak KAKE fare lenfositler etiketlenmesi için kullanılan prosedürler açıklanmaktadır.

Video: The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Antijen Özgül In Vivo</emKAKE Etiketli Hedef Hücreler> Killing Testi

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

İmmünoloji ve Enfeksiyon

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

Karboksi Flöresein sukkinimidil ester (KAKE) Kullanarak İnsan Glioblastoma Yavaş-bölme Hücreleri Tanımlama ve İzolasyonu

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

Tıp

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

Greft Reaktif Bağışıklığı Engelleyen Düzenleyici Makrofajların Fonksiyonel Karakterizasyonu

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

İzolasyon ve murin lenfositlerin aktivasyonu

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Vivo Tahlil antijen spesifik T-hücre Cytolytic işlevi bir aşı modeli kullanılarak tespiti için

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

Fare Retinal Sirkülasyonunda Akış Dinamikleri Çalışmak İçin Eritrositler ve Lökositlerin Floresan Boya Etiketlenmesi

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Proliferasyon ınsan antijeni spesifik CD4+ T hücrelerinin Karboksflorcein Succinimidyl Ester kullanarak ölçülmesini

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

Adjuvan'ın sitotoksik T lenfositler nesil yeteneklerini hızlı Vivo içinde değerlendirilmesi için aşı geliştirme

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation After Stimulation Using CFSE Staining and Flow Cytometry

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
The cell cycle is a universal process of life. During the cell cycle, a cell undergoes several modifications to divide into two daughter cells. This mechanism occurs throughout an organism&#39;s life in response to its needs. Cell divisions and embryonic development produce a full organism from a single-celled zygote. During adulthood, the cell cycle is central to many critical biological processes, such as tissue repairs.
Mechanisms of cell division are tightly controlled events where the cell undergoes stepwise modifications before final division. Cells that are not yet in the cycle are described as being in the Gap 0 (G0) phase. During this stage the cell is considered quiescent. When the cell starts to cycle, four distinct phases are recognized: Gap 1 (G1), Synthesis (S), Gap 2 (G2) and Mitosis (M). G1 phase is a checkpoint for resources needed by the cell for DNA synthesis. Then, S phase occurs, and DNA replication starts, followed by the G2 interphase, another checkpoint that controls all elements necessary to for the cell to divide. Finally, the cell enters mitosis and divides into two daughter cells.
Cell division is a highly informative parameter in many different biological systems. In the field of immunology, analysis of leukocyte proliferation can indicate the mechanism of the immune response. Other domains of investigation also rely on cell cycle analysis. For instance, analysis of the cell cycle during tumor development has improved our understanding of cancer.
Many fluorescent dyes are now available for tracking cell proliferation. These dyes differ in their chemical and spectral properties. Two different classes of dyes exist: protein dyes permanently combine with protein by forming a covalent bond, and membrane dyes stably intercalate within cell membranes via strong hydrophobic associations. In vitro and in vivo studies of immune cell proliferation by flow cytometry are among the most common applications of both classes of cell tracking dyes (1, 2).
CFSE (Carboxyfluorescein succinimidyl ester) is a fluorescent dye that marks dividing cells. Initially, all the cells receive the same amount of dye; dividing cells evenly split the dye they received between their two daughter cells. Consequently, the cell cycle can be followed by the progressive decrease of dye intensity in the cells. CFSE staining is followed by conventional multi-parametric flow cytometry, a high-throughput, fluorescence-based technology that allows phenotypic and functional characterization of cells based on their degree of CFSE staining (3).
In the following experiment, we assess the proliferation of CD4+ and CD8+ T cells in-vitro, following CD3 stimulation, using CFSE staining and flow cytometry.

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur ...

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An In Vitro Assay for Evaluating the Immunomodulatory Impact of Monocytes on Leukocyte Proliferation

Source: Hsu, P., et al. <a href="https://app.jove.com/v/53265">Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs).</a> J. Vis. Exp. (2015)
This video demonstrates an in vitro assay to explore the immunomodulatory influence of mesenchymal stem cells (MSCs) on effector T cell proliferation. The assay includes co-culturing MSCs with PBMCs to induce the monocyte population to acquire an immunomodulatory phenotype, leading to the secretion of interleukin-10 (IL-10). Following incubation with activated CD4+ T cells, the induced monocytes effectively suppress T cell proliferation.

Monositlerin Lökosit Proliferasyonu Üzerindeki İmmünomodülatör Etkisini Değerlendirmek İçin Bir İn Vitro Test

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Density Gradient Isolation of ...

Lenfosit Silahların Yayılmasının Önlenmesi Monitör Carboxyfluorescein diasetat Succinimidyl Ester (KAKE) Kullanımı

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