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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">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., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+intracellular+fluorescent+dye+CFSE+to+monitor+lymphocyte+migration+and+proliferation.">Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation.</a> Current protocols in immunology. , (2009).</li><li>Quah, B. J., Warren, H. S., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dyes+for+lymphocyte+migration+and+proliferation+studies.">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.

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<td>CFDA,SE</td>
<td>&nbsp;</td>
<td>Molecular Probes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>DMSO</td>
<td>&nbsp;</td>
<td>Cambridge Isotope Laboratories Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Lymphocytes</td>
<td>&nbsp;</td>
<td>eg mouse or human</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>RPMI</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>FCS</td>
<td>&nbsp;</td>
<td>SAFC Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>10-15ml conical tubes</td>
<td>&nbsp;</td>
<td>Falcon, Becton Dickinson</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Vortex</td>
<td>&nbsp;</td>
<td>Scientific Industries</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Centrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf (5810 R)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Flow cytometer</td>
<td>&nbsp;</td>
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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 ...

the-use-carboxyfluorescein-diacetate-succinimidyl-ester-cfse-to

Research

JoVE Journal

Immunology and Infection

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

91.6K 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

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

Egzersizle indüklenen Lenfosit Apoptoz belirlenmesi için parmak sopa Kan Örnekleme Metodolojisi

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

İmmünoloji ve Enfeksiyon

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

T6SS aracılı Bakteriyel Rekabet Monitör A Görsel Assay

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

Kullanımı<em&gt; In vivo</em&gt; Görüntüleme Yenidoğanlar Deneysel Fare Sitomegalovirüs Enfeksiyon ilerleme Monitör

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Floresan Proteinleri kullanarak Afrika tripanosom içinde Glycosome Dynamics Monitör

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Hepatit C Virüsü Karşı İnsan laktoferrinin Hücresel Alımı ve İlişkili Antiviral Aktivite Monitör İmmünofloresan

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

Akış Sitometrinin Kullanımı T Hücrelerinde Kromatin Devleti değerlendirmek için

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

T Lenfosit-Yapışma Molekülü Etkileşimleri Çalışmaları Yapışma Altında Kesme Stres Testi

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

Algılama<em&gt; Trypanosoma brucei</emManyetik Aktif Hücre Sorting ve Akış Sitometrisi ile&gt; Varyant Yüzey Glikoprotein Değiştirme

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Bir Hepatit B Virüsü Muhabir Sisteminin Geliştirilmesi Çoğaltma Döngüsü Erken Aşamaları Monitör

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran fare ve insan birincil NK hücreleri Lentiviral iletim verimliliğini artırır

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...