AQUAGAMETE SK 1205 maliyet eylem teşekkür ediyoruz. Bu eser mali AGL201568330-C2-1-R project (MINECO/FEDER) tarafından desteklenmiştir. David G. Valcarce cunta de tarafından finanse edilen Castilla y Leon (EDU1084/2012) ve Fondo sosyal Europeo. Yazar Dr. Ana Riaza ve Stolt deniz çiftlik S.A., Dr Paulino de Paz, Dr Ignacio Martínez Montero ve José Ramón Guiérrez kabul edersiniz. Biz de videografisi için Paula Fernández Colado teşekkür ederim.

Not: Fluorochrome kuluçka ve confocal analiz en az 2-3 h bir kontrol ve bir tedavi örnek alacak. Veri işleme bu zaman hesaplamaya dahil değil. Gerekli malzemeler Malzemeler tablobulunabilir. Bu iletişim kuralı için taze veya cryopreserved sperm uygulanabilir. Solea senegalensis soğuk suda, iş her zaman soğuk koşullarda (4-7 ° C) olarak çoğaltılır bir balık türüdür. Protokol genel görebilmek için bkz: şekil 1 .
1. hazırlık çalışmaları daha önce deneme
<ol><li>1 bir mitokondri leke mM analitik sınıfta DMSO hisse senedi çözüm hazırlamak.</li>
	<li>1 μg/mL ' 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI) hisse senedi çözüm deiyonize su hazır olun.</li>
	<li>Bir 200 mOsm/kg zil çözüm (116 mM NaCl, 2,9 mM KCl, 1.8 mM CaCl2, 5 mM HEPES, pH 7,7) hazırlamak</li>
	<li>4 mM 2 ', 7 '-dichlorodihydrofluorescein diacetate (DCFH-DA) hisse senedi çözüm analitik sınıf metanol içinde hazır olun. Not: stok çözümleri-20 ° C de ihtiyaç kadar aliquots içinde tutun.</li>
	<li>4-7 ° C'de microcentrifuge ayarla</li>
</ol>2. numune hazırlama Fluorochrome kuluçka önce
Not: Pipetting ve resuspending nazik sperm kullanımı tercih edilir.
<ol><li>Eğer cryopreserved hücrelerle çalışma, bir su banyosu Solea senegalensis sperm1040 ° C'de çözdürme için hazır olun. Su banyosu için 7 saman örneklerinde bırakın s.</li>
	<li>Kesmek için makas kullanırken, örnek bir microfuge tüp içine boşaltın ve vasıl 1000 x g, 4-7 ° C için 1 dk santrifüj kapasitesi.</li>
	<li>Seminal plazma veya cryoprotectants ile seminal plazma cryopreserved örnekten boyama protokolüyle engelleyebilir herhangi bir bileşeni atmak için pipetting tarafından kaldırın.</li>
	<li>50-100 μL 4 ° C 200 mOsm/kg zil çözüm hücrelerde resuspend.</li>
	<li>Bir Neubauer veya bir stereoskopik mikroskop altında benzer bir sayım odası ile sperm konsantrasyonu belirlemek.</li>
	<li>1-2 x 106 hücre/mL 0.5 mL 4 ° C'de zil çözüm son hacmi kadar hücre süspansiyon oranında seyreltin.</li>
</ol>3. fluorochrome kuluçka
Not: Fluorochromes, düşük ışık koşullarında, çözümde özellikle ne zaman ele gerekir.
<ol><li>DCFH-DA hisse senedi çözüm 3.125 μL ekleyin (örnekteki son konsantrasyonu: 25 mikron) çalışma Numune dilüsyonu için. 4-7 ° C'de karanlık1040 min için kuluçkaya.</li>
	<li>30 dakika sonra 0.5 μL DAPI hisse senedi çözüm ekleyin (örnekteki son konsantrasyonu: 2 mikron) ve hisse senedi çözüm leke 0.5 μL mitokondri, (örnekteki son konsantrasyonu: 100 nM) örnek. Her iki fluorochromes 10 dk için karanlıkta kuluçkaya.</li>
</ol>4. numune hazırlama için Confocal mikroskobu
<ol><li>Kuluçka süresi sonra santrifüj kapasitesi 1000 x g, 4-7 ° C için 1 dk de hücre süspansiyon.</li>
	<li>Süpernatant dikkatle pipetting tarafından atmak.</li>
	<li>10-20 μL 200 mOsm/kg zil çözüm ekleyin.</li>
	<li>Yer 5 μL konsantre hücre süspansiyon, bir slayt üzerinde bırakın.</li>
	<li>Dikkatle, hazırlık kapak slaytta koymak ve Lehçe ile kapatın. Lehçe kuru olduğunda, hazırlık confocal mikroskopi için hazırdır.</li>
</ol>5. confocal Kur deneme önce
Not: mikroskop bağlı olarak DCFH-DA "yanmış". En iyi koşullarda bir sigara çok değerli örnek ile önce kurun.
<ol><li>Mikroskop, lazerler, kamera ve mikroskop bilgisayarı açın.</li>
	<li>Her fluorochrome uyarma ve emisyon maximums dikkate alarak uyarma lazerler ayarlayın:<ol>
<li>Bir uyarma en çok 358 DAPI için kullanmak nm ve emisyon en fazla 465 nm.</li>
		<li>Mitokondrial leke için bir uyarma en çok 644 kullanmak nm ve emisyon en fazla 662 nm.</li>
		<li>DCFH-DA, bir uyarma en çok 504 kullanmak nm ve emisyon en fazla 525 nm.</li>
	</ol>
</li>
	<li>Sperm hücreleri üzerinde odaklanmak için 25 X amacı ile çalışmaya başlamak. Bu fluorochrome en yüksek yoğunluk raporları, çünkü odak DAPI kanala seçin. Hücreleri odaklandık sonra Solea senegalensis sperm hücreleri boyutu yaklaşık 2 mikron olduğundan, 63 X veya 100 X amacı ayrıntılı bir değerlendirme için kullanabilirsiniz.</li>
	<li>Her kanal için iğne deliği ve sondaj, kazanç ve gerilim en iyi duruma getirme. Aynı şekilde, arka plan azaltmak için dijital ofset ayarlayın. Bir kez tüm parametreleri kullanılan fluorochrome için optimize edilmiş, ayarları kaydedin. Tek bir için bir rutin deney Solea senegalensis sperm hücresi (Bu parametreler her deneyde değişebilir) aşağıdaki kullanılan: 1 ve kazanç gerilimi 750 V iğne deliği.</li>
</ol>6. görüntüleri edinimi
<ol><li>Resim alma için istenen kalite koşullarını seçin. Bit derinliği, görüntü biçimi, hafif levha kalınlığı gibi satın alma ayarlarını tanımlayın ve tek taraflı aydınlatma seçin. Tek bir için bir rutin deney Solea senegalensis sperm hücreleri (Bu parametreler her deneyde değişebilir) aşağıdaki kullandık: çerçeve boyutu 1024 piksel; 16 bit / piksel; tarama hızı 2-4).</li>
	<li>Açın ve başlamak ve alan görüntüsünü almak için tarama alanını Merkezi.</li>
	<li>Kırpma aracını kullanarak, ilgi bölge seçin ve canlı basın.</li>
	<li>Aralığı gösterge aracı yardımıyla arka plan azaltmak için dijital ofset ayarlayın. Her kanal için bunu ve görüntü al.</li>
	<li>Her fluorochrome ve onların birleştirme için bireysel kanalları kalitesini kontrol edin.</li>
</ol>7. bir 3D görüntü Video oluşturma
<ol><li>Z-yığın alma ayarları.<ol>
<li>Yazılımda Z-yığın seçeneği.</li>
		<li>Tek hücre, hücre grubunu veya bölge ilgi seçin ve odaklanır.</li>
		<li>Z-yığın 'First Slice' ve 'Son dilim' seçenekleri ile sınırlandırmak ve aralığı iyi odak yardımıyla 0.2 µm için z adıma kurmak. Bu fluorochrome en yüksek yoğunluk bildirebilir ve kolayca tüm sperm kafa seçebilirsiniz çünkü odaklanmaya DAPI kanalını seçin. Her kanal için en iyi satın alma parametrelerini seçin ve deneme çalıştırın.</li>
	</ol>
</li>
	<li>Z-yığın denemeyi çalıştırmak. Kanalları bölünmüş ve her sinyal hücrelerindeki lokalizasyonu gözlemlemek.</li>
	<li>Z-yığın işlemi.<ol>
<li>Birim render: deneme tamamlandığında, Z-yığın işleme yazılımı seçenekleri seçin ve toplu oluşturun seçeneğini seçin. Adımlar ve döndürme derece (360 °) sayısını seçin. Dosyayı bir video uzantısı ile kaydedin.</li>
		<li>Anaglify: anaglify seçeneği process penceresine. Bu araç bir 3D görüntü split kanallı video oluşturmanızı sağlar.</li>
	</ol>
</li>
</ol>

<ol>
	<li>Amaral, S., 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=Mitochondrial+functionality+and+chemical+compound+action+on+sperm+function.">Mitochondrial functionality and chemical compound action on sperm function.</a> Curr Med Chem. , (2016).</li><li>Morielli, T., O'Flaherty, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+impairs+function+and+increases+redox+protein+modifications+in+human+spermatozoa.">Oxidative stress impairs function and increases redox protein modifications in human spermatozoa.</a> Reproduction. 149 (1), 113-123 (2015).</li><li>G&#252;rler, 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=Effects+of+cryopreservation+on+sperm+viability,+synthesis+of+reactive+oxygen+species,+and+DNA+damage+of+bovine+sperm.">Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species, and DNA damage of bovine sperm.</a> Theriogenology. , (2016).</li><li>Zhu, Z., 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=Vitamin+E+Analogue+Improves+Rabbit+Sperm+Quality+during+the+Process+of+Cryopreservation+through+Its+Antioxidative+Action.">Vitamin E Analogue Improves Rabbit Sperm Quality during the Process of Cryopreservation through Its Antioxidative Action.</a> PLoS One. 10 (12), e0145383 (2015).</li><li>Fang, L., 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=Inhibition+of+ROS+production+through+mitochondria-targeted+antioxidant+and+mitochondrial+uncoupling+increases+post-thaw+sperm+viability+in+yellow+catfish.">Inhibition of ROS production through mitochondria-targeted antioxidant and mitochondrial uncoupling increases post-thaw sperm viability in yellow catfish.</a> Cryobiology. 69 (3), (2014).</li><li>Thomson, L. K., Fleming, S. D., Aitken, R. J., De Iuliis, G. N., Zieschang, J. A., Clark, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation-induced+human+sperm+DNA+damage+is+predominantly+mediated+by+oxidative+stress+rather+than+apoptosis.">Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis.</a> Hum Reprod. 24 (9), 2061-2070 (2009).</li><li>Kim, S. H., Yu, D. H., Kim, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryopreservation+on+phosphatidylserine+translocation,+intracellular+hydrogen+peroxide,+and+DNA+integrity+in+canine+sperm.">Effects of cryopreservation on phosphatidylserine translocation, intracellular hydrogen peroxide, and DNA integrity in canine sperm.</a> Theriogenology. 73 (3), (2010).</li><li>Guthrie, H. D., Welch, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+reactive+oxygen+species+on+sperm+function.">Effects of reactive oxygen species on sperm function.</a> Theriogenology. 78 (8), 1700-1708 (2012).</li><li>Aitken, R. J., Jones, K. T., Robertson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+and+sperm+function--in+sickness+and+in+health.">Reactive oxygen species and sperm function--in sickness and in health.</a> J Androl. 33 (6), (2012).</li><li>Valcarce, D. G., Robles, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+captivity+and+cryopreservation+on+ROS+production+in+Solea+senegalensis+spermatozoa.">Effect of captivity and cryopreservation on ROS production in Solea senegalensis spermatozoa.</a> Reproduction. 152 (5), (2016).</li><li>Gibb, Z., Lambourne, S. R., Aitken, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+paradoxical+relationship+between+stallion+fertility+and+oxidative+stress.">The paradoxical relationship between stallion fertility and oxidative stress.</a> Biol Reprod. 91 (3), (2014).</li><li>Cabrita, E., 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=Factors+enhancing+fish+sperm+quality+and+emerging+tools+for+sperm+analysis.">Factors enhancing fish sperm quality and emerging tools for sperm analysis.</a> Aquaculture. 432, 389-401 (2014).</li></ol>

Yazarlar ifşa gerek yok.

İyi mitokondri sperm motilitesi ve fonksiyon için anahtar organelleri vardır bilinmektedir. Bu organelleri aynı anda doğrudan ROS üretim söz konusu. İlginçtir, ROS kontrollü düzeyde uygun sperm fonksiyonu1için ihtiyaç vardır. Doğurganlık ve oksidatif stres arasında olumlu ilişkiler memeliler11 ' de gösterilen ama sperm kalitesi12aşırı düzeyleri etkiler. Doğru pozitif veya negatif etkisi belirleyici olabilir bir önemli bir fak...

Reaktif oksijen türleri üretim sperm kalitesi ile bağlantılı son zamanlarda1. Ne kadar ROS üretim mitokondri içinde normal bir fizyolojik süreç olarak, ROS üretim aşırı Oksidatif Stresin farklı düzeylerde sperm hücreleri hasara açık bir nedenidir. İnsanlarda, oksidatif stres hareketliliği ve yetenek capacitation2geçmesi için değiştirme erkek infertilitesi ile ilişkilidir; Memelilerde, DNA bütünlük içinde donmuş sperm örnekleri değişikliği de H2O23sentezi için ilgili olmuştur.
Dondurma yetiştiricilik bankacılık gen için ortak bir tekniktir. Bu teknoloji özellikle türlerin Solea senegalensisgibi üreme sorunları ile önemlidir. Piyasada değerli bu türün üreme disfonksiyon bireylerde tutsak Kur eksikliği nedeniyle tarihi gösterir. Bu gerçeği sperm dondurma sperm durumu Yapay döllenme için gerekli kılar. Ancak, dondurma gibi çalışmalar antioksidan takviyesi faydalı etkisini bildirdin bu spermlerin4 için zararlı olabilir oksidatif stres kaynağı olacaktır. ROS inhibisyonu yoluyla mitokondrial hedefli antioksidan bildirildi sperm dondurma sarı yayın balığı5için faydalıdır.
Bu nedenle, ROS seviyelerinde sperm örnekleri bu moleküller sperm hayatta kalma ve üreme8için bir dezavantaj olarak kabul edilmiş, özellikle dondurma6,7 sonra bilmek önemlidir. Ayrıca, ROS dağıtım hücre içinde eğitim potansiyel hasar düzeyi anlaması çok önemli olabilir. Örnek olarak, ROS seviyesinin düşük mitokondri içinde normal ve uyumlu sperm fonksiyonu ile kabul edilebilir ama ROS yüksek düzeyde çekirdek içinde Sperm DNA hasarı belirtileri olabilir. H2O2 mitokondri yayımlanan olabilir ve bir küçük ve ücret daha az molekül9olduğu için çekirdek nüfuz en alakalı ROS biridir. Dichlorofluorescein diacetate (DCFH-DA) özellikle hücre içi peroksit yeşil Floresans yayan ortaya çıkarabilir. Bu makalede, H2O2 hücre içi Yerelleştirme Solea senegalensis sperm confocal mikroskobu kullanılarak tespit için detaylı bir protokol sunulmuştur.

<table border="0" cellpadding="0" cellspacing="0" width="790">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">2&#8242;,7&#8242;-Dichlorodihydrofluorescein diacetate (DCFH-DA)&#160;</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">D6883</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8242;,6-diamidino-2-phenylindole (DAPI)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C1016&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscopy</td>
			<td>Zeiss</td>
			<td>LSM800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover slips</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-541B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO, Analytical Grade</td>
			<td>Sigma-Aldrich</td>
			<td>W387520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol, Analytical Grade</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MitoTrackerDeep Red&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>M22426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge (refrigerated)</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neubauerchamber</td>
			<td>Sigma-Aldrich</td>
			<td>BR717810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>10143562BEF</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of intracellular location of reactive oxygen species in solea senegalensis spermatozoa

Oksidatif stres sperm kalitesini azalan en önemli faktörlerden biridir. Reaktif oksijen türleri (ROS) sperm hücreleri içinde algılamak için etkin iletişim kuralları geliştirme herhangi bir tür yüksek öneme sahip olduğunu, ancak bu yöntemler sık kullanılmayan ve teleost içinde daha az. Dondurma gen bankacılık dahil olmak üzere farklı amaçlar için yararlı bir yöntem su ürünleri içinde ve sperm kullanılabilirlik yıl boyunca garantili. Yordamlar donma/çözülme ROS üretimin neden ve sperm hücrelerine de zarar. Göz önüne alındığında bu potansiyel zarar ROS üretim aşırı onların yerelleştirme bağlı olarak sperm H2O2 algılamak ve hücre içi onun yerelleştirme tarafından confocal mikroskobu değerlendirmek için detaylı bir metodoloji neden sağlanır. Bu amaçla, 3 fluorochromes (2 ', 7 '-Dichlorodihydrofluorescein diacetate (DCFH-DA), bir canlı mitokondri leke ve 4 ', 6-Diamidino-2-phenylindole dihydrochloride (DAPI)) bir arada ortak Yerelleştirme2 H2O değerlendirmek için kullanılır sperm hücreleri çekirdek veya mitokondri Solea senegalesis sperm örnekleri ile.

Confocal mikroskobu teleost sperm hücre içi ROS değerlendirme için ideal bir yöntemdir. Bu çalışmada (şekil 1) sunulan üç fluorochromes (DAPI, bir mitokondri leke ve DCFH-DA) temel araştırma uygulanabilir ve uygulama içinde kullanılan yordamlar geliştirmeye sahip olamaz birçok yararlı bilgi sağlar endüstriyel su ürünleri tesisleri, dondurma iletişim kuralları gibi. Analiz türleri farklı yürütülen hücre içi ROS varlığı ve diğ...

Watch this Scientific Journal Video about Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa at JoVE.com

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

Bu iletişim kuralı H2O2 Yerelleştirme Solea senegalensis sperm hücreleri hassas fluorochrome DCFH-DA ROS, mitokondri için canlı mitokondri leke ve DAPI için çekirdek için kullanma içinde algılamak için detaylı bir metodoloji açıklar görselleştirme, anılan sıraya göre. Protokol taze ya da çözdürülen sperm hücreleri ile 2 saat içinde yapılması için tasarlanmıştır.

Reaktif oksijen türleri Solea Senegalensis sperm hücreleri, hücre içi konumunu değerlendirilmesi

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) ...

evaluation-intracellular-location-reactive-oxygen-species-solea

Research

JoVE Journal

Developmental Biology

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

6.2K Görüntüleme.  University of León. Bu iletişim kuralı H2O2 Yerelleştirme Solea senegalensis sperm hücreleri hassas fluorochrome DCFH-DA ROS, mitokondri için canlı mitokondri leke ve DAPI için çekirdek için kullanma içinde algılamak için detaylı bir metodoloji açıklar görselleştirme, anılan sıraya göre. Protokol taze ya da çözdürülen sperm hücreleri ile 2 saat içinde yapılması için tasarlanmıştır.

Video: Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

İlkel ve kesin hematopoetik ataları insan Pluripotent kök hücrelerden farklılaşma yönetmen

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Gelişim Biyolojisi

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Heterologous IVF, a replacement for homologous IVF, could provide a means to test the sperm fertility potential; to study gamete physiology and early embryo development; and to avoid the use of valuable dolphin oocytes, which are difficult to obtain. Here, we present protocols that have been successfully used to collect and cryopreserve dolphin spermatozoa. The collection of semen is performed by manual stimulation on trained dolphins. Cryopreservation is accomplished using a TRIS egg-yolk based extender with glycerol. In addition, we present a protocol that describes heterologous IVF using dolphin spermatozoa and bovine oocytes and that verifies the hybrid nature of the resulting embryo using PCR. Heterologous fertilization raises questions on fertilization and can be used as a tool to study gamete physiology and early embryo development. In addition, the success of heterologous IVF demonstrates the potential of this technique to test dolphin sperm fertilizing capacity, which is worth further examination.

Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Here, we present protocols that have been successfully used for dolphin spermatozoa collection, cryopreservation, and heterologous IVF performance using bovine oocytes.

Şişe burunlu yunus (Tursiops truncatus) sperm: koleksiyon, dondurma ve kapaklı In Vitro fertilizasyon

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Diseksiyon ve mantar vücut immünfloresan boyama ve yetişkin Drosophila melanogaster beynindeki nöronlar Photoreceptor

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

Yaralanma kaynaklı yaşlanma ve In Vivo kas iskelet yeniden programlama değerlendirilmesi

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

Canlı Drosophila yumurtalık dokusu içinde folikül kök hücre soyundan Imaging hücre içi pH için yöntemleri

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

Caenorhabditis elegans nöron hücre içi taşıma analiz immobilizasyon

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Kromozom segregasyon mitoz sırasında canlı hücre görüntüleme

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. Recent studies have extended the role of cellular senescence to various physiological processes, including development, wound healing, immune surveillance, and age-related tissue dysfunction. Although cell cycle arrest is a critical hallmark of cellular senescence, an increased intracellular reactive oxygen species (ROS) production has also been demonstrated to play an important role in the induction of cellular senescence. In addition, recent studies revealed that senescent cells exhibit potent paracrine activities on neighboring cells and tissues through a senescence-associated secretory phenotype (SASP). The sharp increase in interest regarding therapeutic strategies against cellular senescence emphasizes the need for a precise understanding of senescence mechanisms, including intracellular ROS and the SASP. Here, we describe protocols for quantitatively assessing intracellular ROS levels during H-Ras-induced cellular senescence using ROS-sensitive fluorescent dye and flow cytometry. In addition, we introduce sensitive techniques for the analysis of the induction of mRNA expression and secretion of SASP factors. These protocols can be applied to various cellular senescence models.

Intracellular ROS has been shown to play an important role in the induction of cellular senescence. Here, we describe a sensitive assay for quantifying ROS levels during cellular senescence. We also provide protocols for assessing the senescence-associated secretory phenotype, which reportedly contributes to various age-related dysfunctions.

Reaktif oksijen türleri ve yaşlanma ilişkili salgı fenotip Normal insan fibroblast içinde bir nicel ölçüm onkogen kaynaklı yaşlanma sırasında

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. ...

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy.

Sürgün apikal sürgen farklı bitki türü üzerinden confocal canlı görüntüleme

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses with an egg cell or a central cell. Thus, two independent fertilization events take place almost simultaneously. The fertilized egg cell and central cell develop into zygote and endosperm, respectively. Therefore, precise control of double fertilization is essential for the ensuing seed development. Double fertilization occurs in the female gametophyte (embryo sac), which is deeply hidden and covered with thick ovule and ovary tissues. This pistil tissue construction makes observation and analysis of double fertilization quite difficult and has created the present situation in which many questions regarding the mechanism of double fertilization remain unanswered. For the functional evaluation of a potential candidate for fertilization regulator, phenotypic analysis of fertilization is important. To judge the completion of fertilization in Arabidopsis thaliana, the shapes of fluorescence signals labeling sperm nuclei are used as indicators. A sperm cell that fails to fertilize is indicated by a condensed fluorescence signal outside of the female gametes, whereas a sperm cell that successfully fertilizes is indicated by a decondensed signal due to karyogamy with the female gametes&#8217; nucleus. The method described here provides a tool to determine successful or failed fertilization under in vivo conditions.

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

We demonstrate a method to determine successful or failed fertilization on the basis of sperm nuclear morphology in Arabidopsis double fertilization using an epifluorescence microscope.

Arabidopsis Çift Döllenmede Sperm Nükleer Morfolojisinin İzlenmesi ile Döllenme Durumunun Değerlendirilmesi

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses ...