Bu yazının eleştirel okuma Matsunami laboratuvar üyelerine teşekkür ediyorum.

1. Prosedür: Bu prosedür, 3 gün toplam zaman içinde tamamlandı üç bölümden oluşmaktadır: hücrelerinin transferi, transfeksiyon ve immünhistokimya Hergün yürütülen bir adım. Ilk iki gün içinde transferi ve hücrelerin transfeksiyon laminer akış odasında steril şartlarda yapılmalıdır. 1. Gün: yüzey ifade testi için HEK293T hücre aktarın. <ol><li> Transferi orta (M10):% 10 (hacimce) steril durumda fetal sığır serumu ile takviye asgari temel orta karıştırın. </li><li> Kurmak için gerekli olan plakaların sayısına bağlı olarak, istenen bir confluency 100mm bir kültür plakasına hücreleri büyütün. Büyümüş veya seyrek hücreler önlemek için transfer edilecek hücreler kısmını belirlemek için önemlidir: örneğin,% 100 konfluent 100 mm çanak, yaklaşık% 30 confluency elde etmek için 35 mm çanak yaklaşık% 3 devredebilir ikincisi. </li><li> Önce transfer, hücreler steril laminar akış odası, mont 22 X 22 mm steril poli D lisin (1 mg / ml) ve her biri 35 mm hücre kültürü çanak yer kaplama hücreler için çift tarafı kaplı cam kapak fişleri (Şekil 1 .) 10 dakika - 5 kuruması için çözüm izin verin. Bu süre zarfında, laminer odasında UV kaynak, istenirse kapak fişleri sterilize etmek için açık olabilir. Poly-Ge lisin heterolog hücrelerinin kapak fişleri sopa yardımcı olur. </li><li> Hücreleri aktarmak için, 100mm çanak mevcut medya aspirat, 8 mL steril PBS, aspirat PBS ekleyerek dikkatlice yıkayın ve 3 ml% 0.05 tripsin-EDTA kullanarak hücrelerin trypsinize; hücreleri çanak ayırmak, daha fazla enzimatik reaksiyonu engellemek 5 ml M10 sözlerine ekledi. Hücreleri Karışım ve steril konik tüp Koşul hacim transferi; pelet hücreleri için 5 dakika için 200 gr santrifüj. Hücre pelet zedelemeden, tripsin-EDTA içeren süpernatant aspire. 35 mm yemekleri hücreleri aktarmak için, her yemek için 1 ml M10 ekleyin ve hücreleri ayırmak çiğnemek. Her çanak yeniden süspanse hücrelerinin 1 ml ekleyin. 37 geceleme inkübe hücreleri ° C,% 5 CO 2. <img src="/files/ftp_upload/2405/2405fig1.jpg" alt="Şekil 1" /> Şekil 1. Kat 22 poli D lisin ve her biri 35 mm çanak kaplı yüzü yukarı X 22 mm cam lamelleri. </li></ol> 2. Gün: HEK293T hücrelerinin Transfeksiyon. <ol><li> Aktarıldıktan sonra 18-24 saat, hücre reseptör çalışılmalıdır transfeksiyon için hazır. Reseptör ve 4 önce açıklanan Qiagen Miniprep Kit aşağıdaki protokol kullanarak bakteri kültürleri hazırlanan memeli ekspresyon vektörü PCI klonlanmış RTP1S kullanın. K ölümcül immün kolaylaştırmak için 20 amino asit uzunluğundaki rodopsin epitopu ile etiketlenmiş reseptörleri kullanın. </li><li> Reseptör inşa, 250 ng RTP1S inşa ve 50 ng yeşil floresan protein her transfeksiyon 1000ng kullanın. Üreticileri kılavuzuna göre Lipofectamine 2000, transfeksiyon kullanarak gerçekleştirin. </li></ol> 3. Gün: hücre yüzeyi boyanan reseptörleri görselleştirmek için İmmünositokimya. <ol><li> Başlamadan önce, boyama ve yıkama çözümler hazırlamak ve 4 ° C&#39;ye kadar soğutmak Boyama orta: Minimum Essential Medium, 45 mL; fetal sığır serumu, 5 ml; Hepes (1M), 500 mcL (nihai konsantrasyon 10mM); Sodyum Azid (1,5 M), 500 mcL (son konsantrasyon 15mm). Hepes (1M), 5 ml (son konsantrasyonu 10mM); Sodyum Azid (1,5 M), 5 ml (son konsantrasyon 15mm) HBSS, 500 ml: çözüm yıkayın. Her iki çözüm de yeniden kullanım için 4 ° C&#39;de muhafaza edilebilir. 100 kat boyama orta gerekli antikorları seyreltilmesi ile antikor çözümler hazırlayın. </li><li> 24 saat transfeksiyon hakkında odorant reseptörleri yüzey boyama için immünokimyanın başlatın. Canlı hücre boyama, 4 soğutmalı olmalıdır deney gerçekleştirmek için ° C doldurmak büyük bir buz bulunan ve mümkün olduğunca düz hale getirmek buz yüzey pat, buz üzerinde bir tepsiye yerleştirin ve% 70 ile sprey etanol. Düzgün bir yüzey elde etmek için, bir parafilm parça (tepsinin uzunluğundan daha küçük) ve etanol püskürtülür tepsi yapıştırmayın kesin. Cam kapak, cımbız kullanarak parafilm transfekte hücreler, her seferinde bir hücre yan içeren fişleri aktarın. Katman her kapak kayma (Şekil 2) bir köşesine özenle ekstrüzyon, 100 mcL soğuk birincil boyama çözüm kayma. Katmanlama antikor çözümü ile tüm kapak fişleri sonra, hava çözüm kurumasını önlemek için tepsisi kapağı. 45-60 dakika için birincil inkübasyon yürütmek. </li><li> Birincil inkübasyondan sonra, hızlı bir şekilde, medya içeren orijinal 35mm kültür kaplarına kapak fişleri transfer. Aspire medya ve yemeğin kenarından hafifçe kapak kayma hücreleri kaybını önlemek için 2 mL soğuk yıkama çözeltisi ekleyin. Yıkama solüsyonu aspire edin ve üç yıkar tamamlamak için iki kez tekrarlayın. Sonunda üçüncü yıkama, yıkama solüsyonu aspire yok. </li><li> Ikincil antikor hazırlayın solutiboyama medya ve aynı kasetten yeni yapıştırılmış parafilm tabakası yıkama solüsyonu kapak fişleri transferi. Yine yavaşça 100 mcL birincil inkübasyon gibi köşesinden her kapak kayma tabakası antikor çözüm a&#39;ya. 30 ikincil inkübasyon yürütmek - 45 dakika ve 35 mm orijinal yemekler yıkama solüsyonu transferi geri. Bir kez daha, açıklandığı gibi üç kez kayar kapak yıkayın. </li><li> Aspire son yıkama çözüm yıkayın ve 2 ml% 1 soğutulmuş paraformaldehid eklemek. 15 dakika boyunca buz üzerinde soğutulmuş% 1 paraformaldehid çözüm hücreleri sabitleyin. </li><li> Mount temiz mikroskop cam kapak fişleri hücre yüzü aşağı, montaj reaktif Mowiol kullanarak ve dikkatli tüm hava kabarcıkları (Şekil 3) hariç slaytlar. Bir kez Mowiol hava kurur, yüzey ve hemen aspire distile su ekleyerek kapağını yavaşça fişleri yıkayın. <img src="/files/ftp_upload/2405/2405fig2.jpg" alt="Şekil 2" /> Şekil 2 Katman 100 mcL soğuk birincil boyama çözümü ile her lamel. <img src="/files/ftp_upload/2405/2405fig3.jpg" alt="Şekil 3" /> Şekil 3. temiz cam mikroskop hücre yüzü aşağı Mount lamelleri Mowiol montaj reaktifi kullanılarak kayar. </li></ol> 2. Temsilcisi Sonuçlar: Canlı hücre boyama şaperonlar (protein RTP1S Reseptör taşınması ile bu durumda odorant reseptör Olfr62) (Şekil 4) reseptörlerinin transfeksiyon, hücrelerin yüzeyindeki proteinler görselleştirmek için bir olanak sağlar. Reseptörlerinin hücre yüzeyi boyama boyama noktalı desen (Şekil 4B, içerlek) ile karakterizedir. Boyama, gözlemci, gürültüden gerçek yüzey boyama ayırt etmek zor yapma, uygunsuz yüksek gürültü yürütülen yol açabilir. <img src="/files/ftp_upload/2405/2405fig4.jpg" alt="Şekil 4" /> Şekil 4 şaperonlar ile reseptörlerinin Transfeksiyon canlı hücre boyama kullanarak hücrelerin yüzeyindeki proteinler görselleştirme sağlar. Odorant RTP1S hastabakıcı (B) tek başına reseptör Olfr62 (A) ve Olfr62, transfeksiyon Transfeksiyon. GFP ifade düzeyleri transfeksiyon kontrolü (A &#39;ve B&#39;) olarak hizmet vermektedir.

<ol>
	<li>Buck, L., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multigene+family+may+encode+odorant+receptors:+a+molecular+basis+for+odor+recognition.">A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.</a> Cell. 65, 175-187 (1991).</li><li>Malnic, B., Hirono, J., Sato, T., Buck, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+receptor+codes+for+odors.">Combinatorial receptor codes for odors.</a> Cell. 96, 713-723 (1999).</li><li>Saito, H., Kubota, M., Roberts, R. W., Chi, Q., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RTP+family+members+induce+functional+expression+of+mammalian+odorant+receptors.">RTP family members induce functional expression of mammalian odorant receptors.</a> Cell. 119, 679-691 (2004).</li><li>Zhuang, H., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+cell-surface+expression+and+measuring+activation+of+mammalian+odorant+receptors+in+heterologous+cells.">Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells.</a> Nat Protoc. 3, 1402-1413 (2008).</li></ol>

Her adım, belirgin ve ayırt edici bir yüzey boyama sağlamak için dikkatli bir şekilde yapılmalıdır. Tüm boyama işlemi soğuk (buz) yürütülen olmalıdır ve kapak fişleri hangi tepsiye serilir hücreleri hayatta kalmak sağlamak için kullanmadan önce soğutulması gerekir. Ayrıca fiksasyon yüzey boyama sürecinin sonunda yapılır. Bu fiksasyon birincil ve ikincil antikorlar hücre zarı permeabilize boyama önce iç boyası ile keskin bir kontrast. Hücreleri kurumasını önlemek için kapak fişleri...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cover slip 22 X 22 mm (#1)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>72200-11</td>
<td>&nbsp;</td>
<tr>
<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>16000</td>
<td>&nbsp;</td>
<tr>
<td>Lipofectamine 2000</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11668-019</td>
<td>&nbsp;</td>
<tr>
<td>Minimal essential medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M4655</td>
<td>With L glutamate, Earle&#x2019;s salt and bicarbonate</td>
</tr>
<tr>
<td>Mowiol</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>475904</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>EMS</td>
<td>19208</td>
<td>&nbsp;</td>
<tr>
<td>Hanks Balanced Salt Solution (HBSS) 1X </td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>14025</td>
<td>With calcium chloride and magnesium chloride</td>
</tr>
<tr>
<td>HEPES Buffer Solution</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffer Saline (PBS)</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>21-040-CV</td>
<td>Without calcium and magnesium</td>
</tr>
<tr>
<td>Poly D Lysine Hydrobromide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Azide</td>
<td>&nbsp;</td>
<td>EM Science</td>
<td>SX0299-1</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture dish 35 X 10 mm</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>353801</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA </td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25300</td>
<td>0.05%</td>
</tr>		</tbody>
		</table>
		</div>

assaying surface expression of chemosensory receptors in heterologous cells

Kokuları koku alma duygusunu canlı dünya tarafından kabul edilmektedir. Farelerde koku, uçucu odorant molekülleri bağlamak için öne ve G proteini Gαolf ile kaplin bir hücre içi sinyal ekstraselüler sinyal dönüştürme yaklaşık 1200 G Protein Coupled reseptörleri (GPCRs) 1 bir repertuar ile meydana gelir . Koku reseptörleri bağlayıcı, çeşitli reseptörler bir odorant bağlamak ve bir reseptör derece 2 değişen çeşitli koku bağlamak Kombinatoryal kural takip etmek düşünülmektedir. Biyokimyasal, sinyalizasyon ve ligand bağlanma çalışmaları uygun heterolog hücreleri kullanarak en GPCRs için yürütülen edilmiştir. Ancak, transfeksiyon yüzeye ihracat başarısız beri odorant reseptörlerinin çalışma için heterolog hücrelerinin uzun bir süre engellediği. Saito et al aile şaperonlar co birlikte 3 transfekte heterolog hücrelerinin yüzeyinde, trafik odorant reseptörleri şaperonlar olarak koku duyusal nöronlar ve hareket gelişmiş ifade Protein taşınması tek zarından geçişine Reseptör (RTP) göstermiştir. Reseptör hücre hattı sağlam yüzey ekspresyonu gösterir heterolog hücreleri kullanarak reseptörleri için biyokimyasal testleri yürütmek için bir ilk belirlemeniz gerekir. Bu canlı hücre floresan ekstrasellüler özel ekstrasellüler etki alanı veya bir etiket etiket boyama takip şaperon RTP1S reseptörleri eksprese tarafından test edilebilir. Burada odorant reseptörleri HEK293T hücrelerinin yüzeyinde rahatlıkla tespit etmek için kullanılan olabilir canlı hücre boyama yürütmek için bir protokol göstermektedir. Buna ek olarak, aynı zamanda, diğer kimyasal-duyusal reseptörleri veya GPCRs yüzey ekspresyonu tahlil için kullanılır.

Watch this Scientific Journal Video about Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells at JoVE.com

Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

Burada odorant reseptörleri HEK293T hücrelerinin yüzeyinde rahatlıkla tespit etmek için kullanılan olabilir canlı hücre boyama yürütmek için bir protokol göstermektedir. Buna ek olarak, aynı zamanda, diğer kimyasal-duyusal reseptörleri veya GPCRs yüzey ekspresyonu tahlil için kullanılır.

Heterolog Hücreler kimyasal-duyusal reseptörleri Yüzey İfade Assaying

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors ...

assaying-surface-expression-chemosensory-receptors-heterologous

Research

JoVE Journal

Neuroscience

10.8K Görüntüleme.  Duke University. Burada odorant reseptörleri HEK293T hücrelerinin yüzeyinde rahatlıkla tespit etmek için kullanılan olabilir canlı hücre boyama yürütmek için bir protokol göstermektedir. Buna ek olarak, aynı zamanda, diğer kimyasal-duyusal reseptörleri veya GPCRs yüzey ekspresyonu tahlil için kullanılır.

Video: Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

Sirkadiyen Rhythms Eğitim ve Parametreler Uyku Lokomotor Aktivitesi Assaying Drosophila</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

Nörobilim

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Transgenik kullanarak Assaying β-amiloid Toksisite C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Assaying Lokomotor, Öğrenme ve Hafıza Açıkları. Drosophila Nörodejenerasyon, modeller

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Omurilik Elektrofizyoloji II: Ekstrasellüler Emiş Elektrot İmalatı

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Bozulmamış Duyu Organ Öncü Hücreler Canlı hücre Görüntüleme Drosophila Pupa

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

Kemirgenler ve İnsanlar Burun Koku Kök Hücreler Yalıtımlı

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

Gelişmekte Neuroepithelium Hücre Davranış Yüksek çözünürlüklü Canlı Görüntüleme

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

Comet Assay kullanma Hipokampal Nöronlar DNA Hasarı assaying

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Nöronal Ateşleme Oranlarının Manipülasyon tarafından Astrositik reseptörleri Plastisite Inducing

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

Primer Astrosit Kültürlerinde Proteinlerin Hücre-Yüzey İfadesi ve Endosit Oranının Biyotinleme Yöntemiyle Belirlenmesi

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...