Name: use of lysotracker to detect programmed cell death in embryos and differentiating embryonic stem cells
Uploaded: 2012-10-11T13:00:00
Description: Watch this Scientific Journal Video about Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells at JoVE.com

हम मदद के लिए प्रोटोकॉल संपादन मरिअनी प्रयोगशाला के सदस्यों को धन्यवाद. यह काम एक सीआईआरएम Postdoctoral प्रशिक्षण (JLF) अनुदान, एक सीआईआरएम पुलों इंटर्नशिप (TZTT), रॉबर्ट ई. और मई आर राइट फाउंडेशन (FVM), और दक्षिणी कैलिफोर्निया विश्वविद्यालय (FVM) द्वारा वित्त पोषित किया गया था.

1. माउस भ्रूण की LysoTracker धुंधला <ol><li> पुरुष संवर्धन पिंजरों में युवा महिलाओं (5-6 सप्ताह पुराने) रखने के द्वारा उत्पन्न माउस भ्रूण. एक योनि है कि संभोग का संकेत प्लग की उपस्थिति के लिए निम्नलिखित सुबह पर निगरान...

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sonic+hedgehog-dependent+signaling+relay+regulates+growth+of+diencephalic+and+mesencephalic+primordia+in+the+early+mouse+embryo.">A sonic hedgehog-dependent signaling relay regulates growth of diencephalic and mesencephalic primordia in the early mouse embryo.</a> Development. 129, 4807-4819 (2002).</li><li>Schuldiner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+neuronal+differentiation+of+human+embryonic+stem+cells.">Induced neuronal differentiation of human embryonic stem cells.</a> Brain Res. 913, 201-205 (2001).</li><li>Bain, G., Kitchens, D., Yao, M., Huettner, J. E., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+express+neuronal+properties+in+vitro.">Embryonic stem cells express neuronal properties in vitro.</a> Dev. Biol. 168, 342-357 (1995).</li><li>Dumont, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide-induced+apoptosis+is+CD95-independent,+requires+the+release+of+mitochondria-derived+reactive+oxygen+species+and+the+activation+of+NF-kappaB.">Hydrogen peroxide-induced apoptosis is CD95-independent, requires the release of mitochondria-derived reactive oxygen species and the activation of NF-kappaB.</a> Oncogene. 18, 747-757 (1999).</li><li>Hampton, M. B., Orrenius, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+regulation+of+caspase+activity+by+hydrogen+peroxide:+implications+for+apoptosis.">Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis.</a> FEBS Lett. 414, 552-556 (1997).</li><li>Wood, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+cells+engulf+and+clear+apoptotic+footplate+cells+in+macrophageless+PU.1+null+mouse+embryos.">Mesenchymal cells engulf and clear apoptotic footplate cells in macrophageless PU.1 null mouse embryos.</a> Development. 127, 5245-5252 (2000).</li><li>Scott, R. C., Juhasz, G., Neufeld, T. 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Apoptosis की महत्वपूर्ण पहचान TUNEL परख के साथ डीएनए की क्षति का पता लगाने, कस्पासे गतिविधि का पता लगाने के साथ (mAbs या ZVAD fmk के रूप में इस तरह के एक अणु बंधन के साथ पाया), सेलुलर परिवर्तन के अवलोकन, और phosphatidylserine की प्रस्तु?...

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td></td><td> सूचीपत्र संख्या </td></tr><tr><td> LysoTracker DND-99 लाल </td><td> Invitrogen </td><td> एल 7528 # </td><td></td></tr><tr><td> हैंक्स बीएसएस </td><td> Invitrogen </td><td> 14025-076 </td><td></td></tr><tr><td> Paraformaldehyde </td><td> ईएमडी </td><td> EM-PX0055-3 </td><td></td></tr><tr><td> Vectashield </td><td> VECTOR </td><td> H-1200 </td><td></td></tr><tr><td> DMEM </td><td> Cellgro </td><td> 10-013-CV </td><td></td></tr><tr><td> गैर आवश्यक अमीनो एसिड </td><td> Cellgro </td><td> 25-025-CI </td><td></td></tr><tr><td> सोडियम पाइरूवेट </td><td> Cellgro </td><td> 25-000-CI </td><td></td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30071.02 </td><td></td></tr><tr><td> पेन Strep </td><td> Invitrogen </td><td> 15140-122 </td><td></td></tr><tr><td> ख Mercaptoethanol, 50 मिमी </td><td> Invitrogen </td><td> 21985-023 </td><td></td></tr><tr><td> LabTek-II चैंबर स्लाइड्स (8-अच्छी तरह से) </td><td> Nalge Nunc इंटरनेशनल </td><td> 154534 </td><td></td></tr><tr><td> 0.1 जिलेटिन% </td><td> Millipore </td><td> ES-006-B </td><td></td></tr><tr><td> Dulbecco पीबीएस (डी पीबीएस) </td><td> Cellgro </td><td> 21-031-CV </td><td></td></tr><tr><td></td><td></td><td></td><td> समाधान व्यंजनों 4% Paraformaldehyde 100 मिलीलीटर के लिए: <ol><li> मिश्रण 4 छ paraformaldehyde, 90 मिलीलीटर एच 2 हे, और NaOH (2N NaOH की एक बूंद). paraformaldehyde समाधान में नहीं जाने के लिए जब तक आप कुछ NaOH पीएच बढ़ाने जाएगा. </li><li> हलचल और 60 में गर्मी ° C जब तक सभी पाउडर समाधान (~ 10-20 मिनट) में है. ज़्यादा गरम करना नहीं. </li><li> ~ 10 मिलीलीटर 10x पीबीएस जोड़ें 100 मिलीलीटर की एक अंतिम मात्रा में प्राप्त है. </li><li> सुविधाजनक (~ 10 मिलीलीटर या ~ 40 मिलीलीटर) aliquots में -20 डिग्री सेल्सियस पर स्टोर. </li></ol> चेतावनी: &#39;झालर&#39; फार्म (छर्रों छोटे संकुचित) में Paraformaldehyde कम ख़स्ता है और इसलिए एक डाकू के बाहर मापा जा सकता है. लेकिन आप अभी भी एक सुरक्षात्मक धूल (मुखौटा N95 पहनना चाहिएहैंडलिंग के दौरान कम से कम). EB संस्कृति, मीडिया लिए 500 मिलीलीटर EB मीडिया: <table border="1"><tbody><tr><td> DMEM: </td><td> 404.5 मिलीलीटर </td></tr><tr><td> FBS: </td><td> 75.0 मिलीलीटर </td></tr><tr><td> एल Glutamine: </td><td> 5.0 मिलीलीटर </td></tr><tr><td> पेनिसिलिन / स्ट्रेप्टोमाइसिन: </td><td> 5.0 मिलीलीटर </td></tr><tr><td> गैर आवश्यक अमीनो एसिड: </td><td> 5.0 मिलीलीटर </td></tr><tr><td> सोडियम पाइरूवेट: </td><td> 5.0 मिलीलीटर </td></tr><tr><td> β-Mercaptoethanol: </td><td> 500μl </td></tr></tbody></table></td></tr></tbody></table>

use of lysotracker to detect programmed cell death in embryos and differentiating embryonic stem cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

Watch this Scientific Journal Video about Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells at JoVE.com

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells (Video) | JoVE

हम एक साधारण प्रोटोकॉल मौजूद क्रमादेशित कोशिका मृत्यु का माउस भ्रूण में (PCD) क्षेत्रों और भ्रूण स्टेम सेल (ते) एक उच्च घुलनशील LysoTracker बुलाया डाई का उपयोग संस्कृतियों फर्क कल्पना.

LysoTracker का प्रयोग करने के लिए भ्रूण में क्रमादेशित कोशिका मृत्यु का पता लगाने और भ्रूण स्टेम कोशिकाओं का फर्क

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

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Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Unit 1

Cell Death

SCIENTISTS IN ACTION

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The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth (Video) | JoVE

वीडियो मानव भ्रूण स्टेम सेल कॉलोनी विकास के विश्लेषण बायोइनफॉरमैटिक्स

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors (Video) | JoVE

भ्रूण स्टेम कोशिकाओं के Oligodendrocyte व्यापारियों में भेदभाव

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation (Video) | JoVE

टेराटोमा संरचना द्वारा मानव भ्रूण स्टेम कोशिकाओं की किस्मत मानचित्रण

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...