Bu eser tarafından desteklenmiştir fakülte araştırma hibe Hartford Üniversitesi, üniversite Sanatlar ve Bilimler Dekan'ın ofisinden.

Çalışma sırasında hayvan bakım ve tedavi Ulusal Sağlık Enstitüleri ilkesiyle uyulması, kurumsal kılavuzlarınıza uygun olarak vardı ve Hartford Üniversitesi hayvan kurumsal hayvan bakımı ve kullanımı Komitesi tarafından kabul edildi.
1. hayvanların sürüklenme
Not: 20 haftalık erkek B6129SF2/J fareler çalışmada kullanılır.
<ol><li>Fareler (Standart genel gider beyaz ışık) 12 h ışık için trene binmek / 12 h karanlık döngüsü 2 hafta önce deney için. Not: Burada zeitgeber zaman (ZT) 0 ışıkları ve ZT12, ışıkları tüm diğer çevresel faktörler (Örneğin, yiyecek, su ve oda sıcaklığında) tutarak karşılık gelen sabit 21.</li>
</ol>2. aletleri, kültür orta ve meydan okuma orta hazırlanması
<ol><li>Otoklav forseps ve diseksiyon makası. Buzlu mikroskop slaytlar alüminyum folyo ve basınçlı kap çiftlerini sarın.</li>
	<li>Kültür orta fetal Sığır serum (FBS) ekleyerek RPMI 1640 için % 10 son bir konsantrasyon için hazırlayın. Kültür ortamına aşağıdaki PAMPs ekleyerek 10 mL 50 mL tüpler ortamda meydan hazırlamak (RPMI % 10 ile FBS); LPS (5 µg/mL), ODN1826 (5 µg/mL), ısı-öldürdü Listeria Monositogenez (108 HKLM/mL), veya başka bir PAMP onun önerilen konsantrasyon.</li>
	<li>Sıcak kültür orta ve meydan okuma orta olarak 37 ° C'de bir su banyosu ve yaklaşık 70 mL steril fosfat tamponlu tuz (PBS, pH 7.2) Oda sıcaklığında tutmak.</li>
	<li>Kültür bir 10 mL pipet ve pipet yardım etmek için bir 50 mL tüp ve yer buz üzerinde kullanarak orta 10 mL ekleyin.</li>
	<li>Yaklaşık 30 mL % 70 etanol makas ve forseps mikrobiyal kontaminasyonu önlemek için kabı diseksiyon of a 100 mL kabı ve yer biter ekleyin.</li>
	<li>Lizis arabellek RNA izolasyon için 10 µL β-Mercaptoethanol (altında bir duman hood) ekleyerek arabellek RLT 50 mL tüp içinde her 1 mL hazır olun. Sadece gerekli (yaklaşık 1 x 106 hücreleri oluşur örnek başına 600 µL) olacak miktar olun. Not: Arabellek RLT silika membran için RNA bağlamayı destekler RNA ekstraksiyon kiti bir bileşenidir.</li>
</ol>3. Splenocyte yalıtım ve meydan okuma
<ol><li>Onları kendi özgün kafeste tutma ve 3 L/dak 1 dk. için CO2 durur nefes sonra tedarik devam akış hızında kafesine CO2 ekleyerek narkoz üzerinden belirli zeitgeber zaman fareler ötenazi.</li>
	<li>Servikal yerinden çıkması ile ölüm kafatası tabanında boynun iki tarafında başparmak ve işaret parmağı yerleştirerek onaylayın. Alternatif olarak, kafatası tabanında bir çubuk (Öte yandan kullanarak) hızlı bir şekilde kablolarıylaberaber kuyruk veya servikal vertebra ayrılması kafatası22neden arka bacaklarda Bankası basın.</li>
	<li>Fare gövde ile % 70 etanol ve kağıt havlu ile silin sprey. Üstünde onun sırt ve biraz eğik sağ yan üzerine fareyi getirin. Kürk kes gitsin, anatomi kullanarak, fare sol tarafında hakkında yarım ön ve arka bacakları makas.</li>
	<li>Forseps kullanarak, Karın zarını kapmak ve dikkatle dalak zarar için bir kesik yapın. Forseps ile dalak çıkarın ve Kültür medya buz üzerinde yaklaşık 10 mL içeren bir steril 50 mL tüp içine yerleştirin. Kalan hayvanlar için yineleyin. Not: Dalak bir börülce rengidir ve daha uzun ve böbrek daha düz.</li>
	<li>
Kültür Orta 2 mL ile bir dalak için küçük steril Petri kabına aktarın.
	<ol>
<li>Dalak iki steril buzlu slayt buzlu kısmı arasında taşlama tarafından lunaparkçı. Mümkünse, sorun ve hücreleri ortamda homojenizasyon işlemi sırasında tut.</li>
		<li>Bir kez iyice homojenize damlalıklı kültür splenocytes 50 mL tüp içine 40 µm naylon hücre süzgeç aracılığıyla içeren Orta 2 mL.</li>
		<li>Buzlu slaytları yeni çift, 50 mL tüpler kültür orta ve hücre Süzgeçler kullanarak kalan dalağı için yukarıdaki adımları yineleyin.</li>
	</ol>
</li>
	<li>Her splenocytes için toplam hacmi 10 mL/tüp içeren 50 mL tüpler 8 mL soğuk kültür ortamının ekleyin. Bir hemasitometre kullanarak mililitre başına hücre sayısını belirleyin.</li>
	<li>Yaklaşık 1 x 106 hücreler/iyi 6-şey kültür tabakaları bana ekleyin. Hücreleri de deney için kullanılan farklı PAMPs sayısına karşılık gelen ve bir denetim dahil kuyu sayısını ekleyin. 3 mL kültür ortamının veya meydan okuma orta 3 mL ilgili wells için ekleyin.</li>
	<li>Plakayı %5 CO2 3 h için 37 ° C'de kuluçkaya.</li>
	<li>Hücreleri de P1000 micropipette bağlı bir 1000 µL damlalıklı ucu kullanarak alt kazı. 15 mL tüp için aynı 1.000 µL damlalıklı ucu kullanarak hücreleri içeren orta aktarın.</li>
	<li>Santrifüjü 167 x g oda sıcaklığında 5 min için de üzerinden hücreleri cips. Süpernatant kaldırmak ve hücre Pelet 5 mL de PBS ile yıkayın.</li>
	<li>Hücreleri 167 x g de ikinci kez 5 min için cips, süpernatant kaldırın ve hücreleri parçalayıcı için hücre Pelet lizis arabelleği 600 µL ekleyin. Sonra RNA izolasyon için ilerlemek.</li>
</ol>4. RNA izolasyon ve cDNA sentezi
<ol><li>Splenocytes RNA ekstraksiyon kiti üreticinin yönergelerine uygun kullanarak gelen RNA yalıtmak ve 'isteğe bağlı' sütun üzerinde DNA sindirim gerçekleştirin.</li>
	<li>CDNA sentez önce RNA konsantrasyonu konsantrasyonu en uygun RNA aralığı (en çok 2 µg) içinde olduğunu doğrulamak için bir microvolume spektrofotometre kullanarak cDNA sentez Kit'in belirlemek.</li>
	<li>
Synthesize cDNA her üreticinin yönergelerine uygun ters transkripsiyon seti kullanarak örnekleri için. RNA'ın 10 µL her 20 µL toplam reaksiyon birimindeki örnekleri için kullanın. Ters transkripsiyon (thermocycler çalıştırmak) bitiminde 20 μL Ekle H2O her tepki için.
</li>
	<li>Standart eğri cDNA hazırlamak için 0.5 mL tüp içine birkaç denetim örneklerinden (Örneğin, 5 10 µL toplamda 2 örneklerinden µL) mRNA havuzu için bir P10 veya P20 micropipette kullanarak oluşturun.<ol>
<li>10 µL 2 x transkriptaz ana karışımı ekleyin.</li>
		<li>Ters transkripsiyon bitiminde, standart eğri seyreltme serisinde başlangıç konsantrasyonu ("1") görev yapacak tepki tüp H2O 10 µL ekleyin.</li>
		<li>10 kat seyreltme serisi (1-10-4) 45 µL P100 veya P200 micropipette 10-1, 10-2, 10-3ve 10-4dört 0.5 mL tüpler içine kullanarak su ekleyerek gerçekleştirin.<ol>
<li>5 µL P20 micropipettor, yukarı ve aşağı pipetting tarafından mix kullanarak başlangıç konsantrasyon "1" (10-1) ilk tüp içine eklemek birkaç kez, sonra transfer 5 µL tüp içine 10-1 tüp belirlenmiş 10-2 ve karışımı.</li>
			<li>5 µL P20 micropipette 10-2 tüp 10-3 özel tüp kullanarak transfer ve karıştırın. 5 µL P20 micropipettor 10-3 tüp 10-4 özel tüp kullanarak transfer ve karıştırın.</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. kantitatif polimeraz zincir reaksiyonu (qPCR)
<ol><li>MRNA düzeylerinin göreli Nefelometri qPCR tarafından belirler. İçinde deneysel Kur'u "Nefelometri - göreli standart eğri" ve "TaqMan" Kimya seçin. Değişiklik tepki birime 10 µL. plaka düzenine (Örneğin, hedef gen, standart eğri, muhabir, vb) ilgili bilgileri girin.</li>
	<li>İçerdiği 0.5 µL astar/sonda testin, gen ifade ana karışımı 5 µL, H2O 2 µL ve 2.5 tepki hazırlıkları µL cDNA (10-100 ng).<ol>
<li>Bu standart eğri eklediğinizden emin reaksiyonlar, negatif denetimleri, her tepki performans yinelenen ve hesap olacaktır 2 İlave reaksiyonlar sayısına göre her biri önceki reaktifleri (hariç cDNA) çarparak ana bir karışım hazırlamak Pipetting değişim için.</li>
		<li>Damlalıklı 7.5 µL bir 96-iyi tepki plaka bir micropipette veya bir çok kanallı pipet ile önceden belirlenmiş kuyu içine Ana hazırlanan karışımı. Damlalıklı cDNA bilinmeyenli ve standartları için uygun kuyunun içine 2.5 µL ve H2O negatif kontrol kuyu içine 2.5 µL.</li>
		<li>Çeşitli moleküler saat genler için astar/sonda deneyleri ve aynı zamanda bir tahlil için bir endojen denetimi (Örneğin, β-aktin) içerir.</li>
	</ol>
</li>
	<li>Göreli ifade değerleri belirlemek için her çoğaltma için Ortalama göreli miktarını hesaplamak sonra her örnek için hedef gen için Ortalama göreli miktarı ile β-aktiniçin Ortalama göreli miktarı bölün.</li>
</ol>6. istatistiksel analiz
<ol><li>İstatistiksel analiz yazılımı kullanarak, veri sütun istatistikleri seçeneği altında programa girin. Bir tek yönlü ANOVA saat Gen ifadesinin denetimi karşı PAMP meydan sonra ortalama değerleri arasındaki farklar değerlendirmek için Dunnett'ın öğleden hoc testi ile seçin.</li>
</ol>

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Yazar ifşa etmek hiçbir şey vardır.

Bu protokolde, microvolume spektrofotometre ölçmek ve gen ekspresyonu belirlemede kullanılan RNA saflığı değerlendirmek için kullanılabilir. Nükleik asitler absorbe UV ışığı 260 nm, proteinler genellikle 280 ışığı absorbe bir RNA çıkarma işlemi (Örneğin, fenol) sırasında kullanılan diğer potansiyel kirletici 230 tespit olmakla birlikte nm, nm. Bu nedenle, absorbans (bir) oranında 260/280 değerlendirme tarafından nm (RNA protein) ve 260/230 nm (protein olmayan kirletici için RNA) ...

Neredeyse tüm yönleri için 24 h salınımlarını fizyolojisi ve davranış orchestrates, ana saat memelilerde, suprachiasmatic çekirdek hipotalamus1,2içinde (SCN) yer alır. Bir organizma düzeyinde biyolojik süreçleri düzenleyen ek olarak, ana saatini de periferik hücresel saatler boyunca vücut3,4,5eşitler. En az üç birbirine translasyonel transkripsiyon geri besleme döngüleri moleküler saat makine oluşur ise, çekirdek dönemi (Per1-3), Cryptochrome (Cry1-2), Bmal1oluşur, ve saat genler6,7. Çekirdek moleküler saat doğru zamanlama sürdürmek, yanı sıra bazı yardımcı saat gen ürünleri (Örneğin, Rev erbα ve Dbp) da ifade olmayan genlerine düzenleyen, Yani, kontrollü genler (CCGs)6 saat , 7.
Fonksiyonel moleküler saatler çeşitli bağışıklık dokuları (Örneğin, dalak ve lenf bezleri)8 ve hücreleri (Örneğin, B hücreleri, dendritik hücreler, makrofajlar)8,9tarif edilmistir. Bu hücreler algılamak ve moleküler desenleri patojen ilişkili (PAMPs), doğuştan gelen bağışıklık tanıma reseptörleri Toll benzeri reseptörler (TLR)10gibi ile korunmuş mikrobiyal bileşenleri yanıt. Bugüne kadar 13 fonksiyonel TLRs, hangi bakteri hücre duvarı bileşenleri, kamçılı protein ve mikrobiyal nükleik asitler10gibi mikrobiyal bileşenleri tanımak tarif edilmistir. PAMP, lipopolysaccharide (LPS), gram-negatif bakteri TLR4 tarafından tanınan bir hücre duvarı bileşeni sirkadiyen ritim, organizma ve moleküler düzeyde değiştirmek için gösterilmiştir. Örneğin, LPS vivo içinde meydan etkinlik fareler11 tarafından ölçülen fotosentez benzeri faz gecikmeler indüklenen ve indirimli saat gen ekspresyonu SCN (sahne) ve in situ hibridizasyon ve kantitatif PCR ile belirlendiği gibi karaciğer yol açtı, sırasıyla, içinde12sıçanlar. Sonra bir vivo içinde sorun LPS ile qPCR ile ölçülen birkaç saat genlerin değişmiş ifade insan periferik kan lökosit13 ve deri altı yağ dokusu14 analizi ortaya koydu. Son olarak, insan makrofajlar ve fare peritoneal makrofajlar, ayrıca yol açtı ex vivo LPS sorunları qPCR14tarafından ölçülen saat ifade değiştirdi.
Burada, biz PAMPs LPS, (sentetik oligonucleotides içeren bazlar CpG motifler) ODN1826, etkisini değerlendirmek için bir protokol tanımlamak ve TLR4, TLR9 ve TLR2, tarafından tanınan ısı öldürdü Listeria Monositogenez (HKLM), sırasıyla, Moleküler saat Gen ifadesinin içinde fare splenocytes. Protokol fare splenektomi, splenocyte yalıtım ve meydan okuma, RNA ayıklama, cDNA sentezi ve qPCR birkaç saat genlerin ifade değerlendirmek için içerir. Bu iletişim kuralı çok az hayvan ile bağışıklık hücreleri çok sayıda zamanında edinimi için izin verir veya daha sonra olabilir hücresel manipülasyon ex vivo çeşitli PAMPs ile meydan. Moleküler saat bağışıklık yanıtı8,15,16, çeşitli açılardan modüle gösterilmiştir, bu nedenle, moleküler saat çalışmasının büyük olasılıkla uygun zamana bağımlı varyasyonu zarar bağışıklık yanıtı. Sirkadiyen ritim kesintileri ciddi patolojiler17,18,19,20' ye neden olabilir beri Ayrıca, araştırmacılar splenocytes geniş bir yelpazede meydan okumak için ilgi olabilir moleküller ve saat onların etkisi değerlendirmek.

<table><tbody><tr><td>Frosted slides</td><td>Fisher</td><td>12-550-343</td><td></td></tr><tr><td>Cell strainers</td><td>Fisher</td><td>22363547</td><td></td></tr><tr><td>Lipopolysaccharide&amp;nbsp;</td><td>InvivoGen</td><td>ltrl-eklps</td><td></td></tr><tr><td>ODN1826</td><td>InvivoGen</td><td>Tlrl-1826-1</td><td></td></tr><tr><td>HKLM</td><td>InvivoGen</td><td>Tlrl-hklm</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>20012-043</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104 or 74106</td><td></td></tr><tr><td>RNase-Free DNase Set</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>6-well cell culture plate</td><td>Denville</td><td>T1006</td><td></td></tr><tr><td>50 mL tubes</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>15 mL tubes</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription Kit</td><td>ThermoFisher</td><td>4368814</td><td></td></tr><tr><td>TaqMan Gene Expression Assays b-actin</td><td>ThermoFisher</td><td>Mm00607939_s1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Per2</td><td>ThermoFisher</td><td>Mm00478113_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Rev-erba</td><td>ThermoFisher</td><td>Mm00520708_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Bmal1</td><td>ThermoFisher</td><td>Mm00500226_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Dbp</td><td>ThermoFisher</td><td>Mm00497539_m1</td><td></td></tr><tr><td>qPCR machine StepOnePlus</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>ThermoFisher</td><td>4369016</td><td></td></tr><tr><td>MicroAmp Fast 96-well reaction plate (0.1 mL)</td><td>ThermoFisher</td><td>4346907</td><td></td></tr><tr><td>Statistical Analysis Software</td><td>Prism 7.0a</td><td></td><td></td></tr></tbody></table>

the use of mouse splenocytes to assess pathogen-associated molecular pattern influence on clock gene expression

Davranış-gen ekspresyonu sirkadiyen ritim fizyolojisi neredeyse tüm yönlerini düzenleyen. Burada, fare splenocytes moleküler desenleri (PAMPs) patojen ilişkili lipopolysaccharide (LPS), ODN1826 ve ısı öldürdü Listeria Monositogenez meydan ve moleküler üzerindeki etkileri sirkadiyen incelemek için bir metodoloji mevcut saat. Daha önce çalışmalar LPS etkisi a değişiklik-in vivo ve ex vivo yaklaşımlardan modelleri (Örneğin, fare, sıçan ve insan) bir ürün yelpazesine kullanarak moleküler saat inceleyerek üzerinde odaklanmıştır. Bu iletişim kuralı yalıtım ve saat gen ifade sonrası meydan kantitatif PCR ile değerlendirmek için meydan okuma splenocytes yanı sıra metodoloji açıklanmaktadır. Bu yaklaşım sadece etkisi mikrobiyal bileşenleri ifade saatin değiştirebilir moleküler saat diğer moleküller de ama değerlendirmek için kullanılabilir. Bu yaklaşım ayrı nasıl etkilediği PAMP Toll benzeri reseptör etkileşim saat ifade moleküler mekanizması kızdırmak için yararlanılabilir.

Fareler ZT13 kurban edildi, splenocytes izole edildi ve ex vivo PAMPs LPS, ODN1826 veya HKLM meydan. 3 h sonra RNA izole edildi ve qPCR saat, Per2, Dbp ve Rev-erbα tartışmasız denetim hücrelere kıyasla moleküler saat genlerin göreli ifade düzeylerini değerlendirmek için kullanılır. PAMP meydan sonra saat ifade düzeyleri kontrol hücreleri (Şekil 1A) ifadesinde önemli ölç?...

Watch this Scientific Journal Video about The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression at JoVE.com

The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

Bu protokolü moleküler saat gen ekspresyonu alter patojen ilişkili moleküler desenleri bulmak için fare splenocytes kullanarak bir teknik anlatılmaktadır.

Saat Gen ifadesinin etkisi moleküler desen patojen ilişkili değerlendirmek için fare Splenocytes kullanımı

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse ...

the-use-mouse-splenocytes-to-assess-pathogen-associated-molecular

Research

JoVE Journal

Genetics

7.4K Görüntüleme.  University of Hartford. Bu protokolü moleküler saat gen ekspresyonu alter patojen ilişkili moleküler desenleri bulmak için fare splenocytes kullanarak bir teknik anlatılmaktadır.

Video: The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

İnsan Primer Hücre Kültürleri içinde Sirkadyen Saat Gen İfade ve Hormon salgısını Paralel Ölçümü

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Genetik

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

Vitro Sirkadiyen ritim meme epitel hücreleri ayırdetmek için bioluminescence tahlil

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

Detaylı Mekansal İfade Haritalar Dinamik İfade Verilerinin Zamansal Sipariş

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Gelişim Biyolojisi

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

Biyoluminesen Bakteriler Kullanılarak Gerçek Zamanda Bir Akut Fare Pnömoni Modelinde Pnömokok virulans faktörlerinin etkisi aşağıdaki

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

İmmünoloji ve Enfeksiyon

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Lokal ve sistemik Yer de patojen kaynaklı kronik enflamasyon Fare Modeli

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal allophycocyanin (APC) fluorescence intensity, are conjugated to antibodies specific to a particular analyte. Next, a selected panel of defined capture bead sets is incubated with a biological sample containing target analytes specific to the capture antibodies. A biotinylated detection antibody cocktail is added, which leads to the formation of capture bead-analyte-detection antibody sandwiches.
Finally, streptavidin-phycoerythrin (SA-PE) is added, which binds to biotinylated detection antibodies, providing fluorescent signal intensities in proportion to the amount of bound analyte. The PE fluorescent signal of analyte-specific beads regions is quantified using flow cytometry, and the concentrations of particular analytes are determined using data analysis software and the standard curve generated in the assay.
In this experiment, we use a mouse T helper cytokine panel to simultaneously quantify the concentration of 13 separate cytokine targets in tissue culture supernatants collected from mouse splenocytes cultured under various stimulatory conditions.

This protocol describes the quantification of multiple cytokine targets simultaneously in tissue culture supernatants collected from stimulated mouse splenocytes using multiplex bead based immunoassay platform and a flow cytometer.

Multiplex sitokin Immunoassay sitometrik boncuk tabanlı platformu kullanarak uyarılmış fare Splenocytes profil oluşturma

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Bakteriyel Pathogen sonda için hücresel ve Organismic düzeyi ana bilgisayar yanıt için kullanma

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 &#215; 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

Vivo Sirkadiyen saat gen ekspresyonu floresans gazetecilere kullanarak fare Suprachiasmatic çekirdeği olarak izlenmesi

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

Nörobilim

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

Listeria monocytogenes enfeksiyonu sonrasında Mouse dalağında In situ Interferon gama üretiminin görüntülenmesi

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.