CQS receives a fellowship from the LOEWE Excellence Cluster for Integrative Fungal Research (IPF). The author would like to thank Ida J. van der Klei for the P. chrysogenum strains used in this work and Andreas S. Reichert for the gift of rapamycin.

P. hazırlanması 1. chrysogenum Açlık Deneyler için <ol><li> P. Eğer Çevrede chrysogenum suşu pirinç (&#39;yeşil pirinç&#39;) tutulur, 1.5 ml mikrosantrifüj tüp içine miselyumu sporlanan ile kaplı 2-3 pirinç tanelerini yerleştirin. 500 ul YGG (10 g / l KCI, 20 g / l glukoz, 10 g / l maya azot baz, 5 g / l K 2 HPO 4, 20 g / l maya ekstresi) ile doldurun. <ol><li> Sporlar etkili bir pirinç ayrılacak şekilde 30 saniye için tüp karıştırın. </li><li> Oda sıcaklığında 1 gün (örneğin, 20 ila 25 ° C) tüp inkübe edin. </li><li> Steril musluk suyu bu spor süspansiyonu, bir 1/50 seyreltme hazırlanır, iyice karıştırın. </li></ol></li><li> P. Eğer Çevrede chrysogenum suşu bir agar plaka (örneğin, YGG agar) tutulur, 400 ul steril musluk suyu ve yaklaşık 100 mg gl içeren 1.5 ml mikrosantrifüj tüp içine misel küçük parçalar aktarmaksteril bir kürdan veya pipet kullanarak eşek boncuk. <ol><li> Vortex 2 dakika süreyle tüp böylece misel parçalanmış olur. Hemen tüp içeriğini kullanın. </li></ol></li></ol> P. Yetiştiriciliği 2. Hazırlık Adımları Açlık Pedleri üzerinde chrysogenum <ol><li> Bir ısıtma plakası üzerinde açma ve yaklaşık 60 ° C&#39;ye ayarlayın. </li><li> Bir mikrodalga fırında pişirme çözümü ile musluk suyu ml 100&#39;de agaroz 2 gr eritin. Bu çözüm ısıttıktan sonra tamamen açıktır özen gösterin. Değilse agaroz tamamen eriyene kadar, tekrar pişirin. Agaroz çözüm kullanılmadan önce yaklaşık 60 ° C&#39;ye kadar soğumasını bekleyin. </li><li> 400 ul steril musluk her (ek bileşikler eklenmiştir) su ya da (örneklerin sayısına bağlı olarak 2-4) 1.5 ml mikrosantrifüj tüpleri Dolgu 400 x ul (adım 2.5 bileşiği çözeltisi ul x ilaveli) ve koyun en az 1 dakika için ısıtma plakası üzerine bu yüzden water içinde ısınıyor. </li><li> En az 1 dakika için ısıtma plakası üzerine bir merkezi boşluğu içeren mikroskop slayt yerleştirin. </li><li> 1.5 ml mikrosantrifüj tüpleri ve girdap kısaca çözeltisine agaroz 400 ul% 2 ekleyin. Eğer istenirse bu adımdan sonra ilave bileşikler (yani, 1 uM rapamisin) ekleyin. Eğer bu yapılırsa, girdap her ek madde tüp pipetle kısaca sonra. Erken katılaşma önlemek için ısıtma masaya geri tüpleri koyun. NOT: mikrosantrifüj tüpü içinde toplam hacim 800 ul olmalıdır. </li></ol> Açlık Pedleri İçeren Mikroskop Slaytlar 3. Hazırlık <ol><li> (Hala ısıtma plakası üzerinde yer almaktadır) mikroskop lamı boşluğuna agaroz çözeltisi Pipet 140 ul. Mümkünse kabarcıkları yapmaktan kaçınmaya çalışın. </li><li> Hemen agaroz çözeltisi üzerine bir kapak kayma yerleştirin. Not: Bu, düz bir yüzeye yol açacaktır. </li><li> Mikro koyYaklaşık 4 dakika laboratuar tezgah üzerine kapsam slayt agaroz ped katılaşmaya izin. </li><li> Dikkatlice başparmak veya işaret parmağı yardımı ile kaymasını agaroz ped lamel çıkarın (kirlenmeyi önlemek için eldiven giymek!). </li><li> Kurumasını önlemek için bir ıslak odasına ped ile mikroskop lamı yerleştirin. Öneri: hala pipet uçları tutmak için ekleme sahip boş bir ipucu kutusunu kullanın. Alt tutulmasını sağlamak için, kutu, steril su eklenir. Insert üzerine mikroskop slaytlar yerleştirin ve kutusunu kapatın. </li></ol> 4. P. ile Açlık Pedleri aşılamak chrysogenum Kuluçka <ol><li> 1/50 spor (kültürler pirinç yetiştirilen olsaydı) seyreltme veya açlık pad merkezine üzerine (kültürler bir agar plaka üzerinde yetiştirilen olsaydı) misel parçalarını içeren sulandırılmamış çözeltinin 5 ul Pipet 5 ul. </li><li> Bu şekilde hizmet vermektedir (ıslak odasına kapatın ve bir plastik torba içinde sarınaçlık yastıkları kuruma karşı ek koruma). Çok aksi aşılama damla rahatsız olacak, çünkü kutusunu taşımak için dikkatli olun. </li><li> Örnekleri analiz etmek için en az 20 saat boyunca oda sıcaklığında sarılmış kutusunu saklayın. </li></ol> Örneklerinde 5. mikroskobik analizi <ol><li> Inkübasyon 20 saat sonra numuneler mikroskobik analiz için hazır; Kuru kağıt mendil üzerine mikroskop lamı koyun (alt ıslak olma eğilimindedir). </li><li> Açlık ped üzerine küçük bir su hacmi (yaklaşık 50 ul) pipetle. Ped üzerine bir lamel koyun ve fazla suyunu sıkın. Bir kağıt mendil ile fazla suyun çıkarın. </li><li> Floresan mikroskop gözlem masaya mikroskop lamı koyun. Misel büyüme bir bakış elde etmek için, bir 20X objektif kullanın. Hif GFP lokalizasyonu incelemek için kullanın 63X veya 100X hedefleri. Için numune analiz yok numune kademeli kuruma önlemek içinuzun 15 dakika ıslak odasına geri koymadan önce. </li><li> (40 saat ve büyüme 60 saat sonra, örneğin) düzenli aralıklarla 5.3 tekrarlayın. NOT: Numune ıslak odasına geri konabilir. O Miseliyum büyümeyi etkilemez gibi lamel kaldırmak için gerekli değildir. </li></ol>

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	<li>Fischer, F., Hamann, A., Osiewacz, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+quality+control:+an+integrated+network+of+pathways.">Mitochondrial quality control: an integrated network of pathways.</a> Trends Biochem. Sci. 37 (7), 284-292 (2012).</li><li>Pollack, J. K., Harris, S. D., Marten, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+filamentous+fungi.">Autophagy in filamentous fungi.</a> Fungal Genet. Biol. 46 (1), 1-8 (2009).</li><li>Scheckhuber, C. Q., Osiewacz, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Podospora+anserina:+a+model+organism+to+study+mechanisms+of+healthy+ageing.">Podospora anserina: a model organism to study mechanisms of healthy ageing.</a> Mol. Genet. Genomics. 280 (5), 365-374 (2008).</li><li>Pinan-Lucarr&#233;, B., Iraqui, I., Clav&#233;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Podospora+anserina+target+of+rapamycin.">Podospora anserina target of rapamycin.</a> Curr. Genet. 50 (1), 23-31 (2006).</li><li>Kikuma, T., Ohneda, M., Arioka, M., Kitamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+the+ATG8+homologue+Aoatg8+and+role+of+autophagy+in+differentiation+and+germination+in+Aspergillus+oryzae.">Functional analysis of the ATG8 homologue Aoatg8 and role of autophagy in differentiation and germination in Aspergillus oryzae.</a> Eukaryot. Cell. 5 (8), 1328-1336 (2006).</li><li>Pinan-Lucarr&#233;, B., Balguerie, A., Clav&#233;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+cell+death+in+Podospora+autophagy+mutants.">Accelerated cell death in Podospora autophagy mutants.</a> Eukaryot. Cell. 4 (11), 1765-1774 (2005).</li><li>Veneault-Fourrey, C., Barooah, M., Egan, M., Wakley, G., Talbot, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagic+fungal+cell+death+is+necessary+for+infection+by+the+rice+blast+fungus.">Autophagic fungal cell death is necessary for infection by the rice blast fungus.</a> Science. 312 (5773), 580-583 (2006).</li><li>Bartoszewska, M., Kiel, J. A., Bovenberg, R. A., Veenhuis, M., van der Klei, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+deficiency+promotes+beta-lactam+production+in+Penicillium+chrysogenum.">Autophagy deficiency promotes beta-lactam production in Penicillium chrysogenum.</a> Appl. Environ. Microbiol. 77 (4), 1413-1422 (2011).</li><li>Meijer, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisomes+are+required+for+efficient+penicillin+biosynthesis+in+Penicillium+chrysogenum.">Peroxisomes are required for efficient penicillin biosynthesis in Penicillium chrysogenum.</a> Appl. Environ. Microbiol. 76 (17), 5702-5709 (2010).</li><li>Cubitt, A. B., Heim, R., Adams, S. R., Boyd, A. E., Gross, L. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding,+improving+and+using+green+fluorescent+proteins.">Understanding, improving and using green fluorescent proteins.</a> Trends Biochem. Sci. 20 (11), 448-455 (1995).</li><li>Jung, C. H., Ro, S. H., Cao, J., Otto, N. M., Kim, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTOR+regulation+of+autophagy.">mTOR regulation of autophagy.</a> FEBS Lett. 584 (7), 1287-1295 (2010).</li><li>Hickey, P. C., Read, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+living+cells+of+Aspergillus+in+vitro.">Imaging living cells of Aspergillus in vitro.</a> Med. Mycol. 47, S110-S119 (2009).</li></ol>

The author has nothing to disclose.

Burada sunulan yöntem, P. otofaji uygun ve tekrarlanabilir çalışma sağlar chrysogenum. Örneğin, bu mantar ya da değil Otofaji tepkisini modüle edebilen olup çeşitli bileşiklerin etkinliğini taramak için kullanılabilir. rapamisin ile sonuçlar Devri sinyallemesinin engellenmesi P. otofaji belirgin bir indüksiyonuna neden olduğunu göstermektedir chrysogenum, diğer organizmalar 11 kanıtlanabildiği. Daha karmaşık mikroskoplar?...

İpliksi mantar gelişme işlemine çalışma için mükemmel bir model sistemleridir. Bunlar ucuz ekimi, döl ve genetik erişilebilirlik yüksek sayılar gibi birçok deneysel faydaları sunuyoruz. son nokta, çeşitli hücresel mekanizmalar için çok-çok uncharacterized genlerin önemini araştıran izin Transformantların yapımı için özellikle önemlidir. İpliksi mantar fazlası ve / veya işlevsel hücre bileşenlerinin çıkarılması için mitokondriyal bütünlüğü ve Otofaji sağlamak ve sürdürmek için çeşitli elemanlar ve anormal protein, mitokondriyal dinamikleri indirgenmesi için proteaz aktivitesi hücresel kalite kontrol yollarının mekanizmaların açıklanması için etkili olmuştur açlık 1,2,3 zamanlarında hücre canlılığı. Ipliksi mantarlar 2 Otofaji çalışma için uygun bir çok deneysel teknik vardır: vaküol (i) inceleme ıproteazlar, transmisyon elektron mikroskopisi 4 tarafından önlendiği zaman f de yoğun otofajik organları içeren, (ii) flüoresan boya monodansyl kadavenn kullanılarak floresan mikroskop 5,6 ve asidifiye autophagosomal yapıları (iii) algılama ile GFP Atg8 odak izleyerek autophagosomes görselleştirme 7. Burada, otofaji çalışmaları için penicillium chrysogenum büyüyen yeni bir yöntem sunulmaktadır. Ana unsur sadece agaroz sterilize musluk suyunda çözünmüş 1% oluşur &#39;açlık pedi&#39; olduğunu. Ek bileşikler (örneğin, stres, temizleyiciler, otofaji modülatörleri) sürece otomatik floresan göstermez altlığınızda ilave edilebilir. ped sığ merkezi boşluğu ihtiva mikroskop slaytlar yer almaktadır. Bir spor süspansiyonu ile ya da küçük miselyum fragmanları ile aşılanır Bu ped. ilgi suşu sporulat başarısız olursa ikinci tavsiye ediliretkin bir şekilde, e (örneğin, Δ atg1 suşları 8). slaytlar numunenin kuruma önlemek için (bunlar kolayca boş pipet ucu kutuları kullanılarak inşa edilebilir) ve oda sıcaklığında inkübe ıslak odalarında konumlandırılmış. P. chrysogenum, bu koşullar altında bir kaç gün için büyümeye edebilmektedir. Otofaji mantar Otofaji için olumlu bir belirtecidir vakuolar genişleme ile mikroskobik edilebilir. Bu katkı, bir P. chrysogenum soyu (Wisconsin 54-1255) C-terminal &#39;SKL&#39; dizisi 9 ile peroksisomlara hedeflenen yeşil floresan proteini oluşturan kullanılır. Bu nedenle, peroksizom bozulmasını izlemek mümkündür. Uygun lokalizasyon sinyallerini kullanarak, aynı zamanda hücrenin (örneğin, mitokondriya) &#39;in diğer bölmeleri etiket ve bozunmasını analiz etmek mümkündür. P. veri rağmen chrysogenum Ws54-1255 (GFP-SKL) kesinlikle mümkün, burada sunulmuşturDiğer ipliksi mantarlar için de &#39;açlık ped&#39; yöntemi kullanmak (örneğin, Neurospora crassa, Sordaria macrospora, Aspergillus türleri, vb).

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			<td style="width:71px;">Company</td>
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			<td style="width:167px;">Comments/Description</td>
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			<td height="105" style="height:105px;width:216px;">Microscope slides with central cavity</td>
			<td style="width:71px;">Carl Roth GmbH, Karlsruhe, Germany</td>
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			<td>These can be used multiple times after cleaning.</td>
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			<td height="105" style="height:105px;width:216px;">Glass beads</td>
			<td style="width:71px;">Carl Roth GmbH, Karlsruhe, Germany</td>
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			<td>Diameter: 0.25 - 0.50 mm</td>
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analysis of autophagy in penicillium chrysogenum by using starvation pads in combination with fluorescence microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

P. peroksizom bozulması yukarıda ayrıntılı protokol kullanılmasını göstermek için chrysogenum suşu Ws54-1255 (GFP-SKL) analiz edildi. Bu gerginlik GFP-SKL genellikle peroksizom 9 aktarılır. Örnek floresan mikroskobu ile analiz edildiğinde, bu çok sayıda küresel şekiller görünümüne yol açar. Otofaji oluşursa, vakuolleri büyütmek. GFP-SKL otofaji (pexophagy) tarafından vakuollerde dahil olur. Nedeniyle GFP vakuol proteazlar tarafından bozulmaya dirençli olduğu ge...

Watch this Scientific Journal Video about Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy at JoVE.com

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

Otofaji Analiz<em&gt; Penicillium chrysogenum</em&gt; Floresan Mikroskopi ile Kombinasyon Açlık Pedleri kullanarak

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

analysis-autophagy-penicillium-chrysogenum-using-starvation-pads

Research

JoVE Journal

Biology

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

8.5K Görüntüleme.  Senckenberg Research Institute. A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

Video: Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Zaman atlamalı Görüntüleme siRNA Transfeksiyon sonra Mitoz

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Biyoloji

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Gelişmiş 3D Floresan Mikroskobu kullanılarak Otofaji Kantitatif Analiz

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Floresan kullanarak mRNA Moleküllerin Görselleştirme ve Analiz<em&gt; Yerinde</emIn&gt; Hibridizasyon<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fekal Glukokortikoid Analizi: Tek tırnaklılarda in Non-invaziv Adrenal İzleme

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Floresan Mikroskopi ile Stromule Frekansı görselleştirme

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Activating Autophagy by Aerobic Exercise in Mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

Farelerde Aerobik Egzersiz ile Autophagy etkinleştirme

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

VirusMapper Süper çözünürlüklü Mikroskopi Açık kaynak Tek parçacık Analizi

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Floresans canlı hücre mikroskobu ile mitotik ve meiotic Fission Maya nükleer dinamiklerinin incelenmesi

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

Toplam İç Yansıma Floresan Mikroskobu Ile Canlı Hücrelerdeki Hücre Yüzey Reseptörlerinin Oligomerizasyon Dinamiği Sayı ve Parlaklık Analizi ile Birlikte

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...