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 Video at JoVE.com

Мы благодарим членов Мариани лаборатории для помощи редактирования протокола. Эта работа финансировалась CIRM Докторантура Обучение Грант (JLF), стажировка CIRM BRIDGES (TZTT), Роберт Э. и мае Р. Райт Foundation (МКО) и Университета Южной Калифорнии (МКО).

1. LysoTracker Окрашивание эмбрионов мыши <ol><li> Создание эмбрионов мыши, поместив молодых (5-6 недель) женщин в мужских клетках студ. Монитор на следующий утрам для появления вагинального вилку о том, что спаривание произошло. Если женщина беременна, в полдень в этот день называют 0,5 DPC (дней пос...

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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. 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Важные признаки апоптоза включают обнаружение повреждений ДНК с анализом TUNEL, наряду с обнаружением активности каспазы (обнаружен с МАТ или связывающей молекулы, такие как ZVAD-FMK), наблюдение клеточные изменения, и представление фосфатидилсерина (PS ) на поверхности мембраны (обнаружен а?...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td></td><td> Номер в каталоге </td></tr><tr><td> LysoTracker Красной DND-99 </td><td> Invitrogen </td><td> # L-7528 </td><td></td></tr><tr><td> Хэнкс BSS </td><td> Invitrogen </td><td> 14025-076 </td><td></td></tr><tr><td> Параформальдегид </td><td> EMD </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> Номера для незаменимых аминокислот </td><td> Cellgro </td><td> 25-025-ДИ </td><td></td></tr><tr><td> Пирувата натрия </td><td> Cellgro </td><td> 25-000-ДИ </td><td></td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30071.02 </td><td></td></tr><tr><td> Pen-Strep </td><td> Invitrogen </td><td> 15140-122 </td><td></td></tr><tr><td> б-меркаптоэтанола, 50 мМ </td><td> Invitrogen </td><td> 21985-023 </td><td></td></tr><tr><td> LabTek-II палаты слайды (8-а) </td><td> Nalge Nunc Международного </td><td> 154534 </td><td></td></tr><tr><td> 0,1% желатина </td><td> Millipore </td><td> ES-006-B </td><td></td></tr><tr><td> Дульбекко PBS (D-PBS) </td><td> Cellgro </td><td> 21-031-CV </td><td></td></tr><tr><td></td><td></td><td></td><td> Решение рецепты 4% параформальдегида На 100 мл: <ol><li> Смешайте 4 г параформальдегида, 90 мл H 2 O и NaOH (капли 2N NaOH). Параформальдегид не буду вдаваться в решение пока не будут добавлены некоторые NaOH для увеличения рН. </li><li> Перемешать и тепла при температуре 60 ° C, пока все порошка в раствор (~ 10-20 мин.) Не перегревать. </li><li> Добавить ~ 10 мл PBS 10x для достижения конечного объема 100 мл. </li><li> Хранить при -20 ° C в удобное (~ 10 мл или ~ 40 мл) аликвоты. </li></ol> ВНИМАНИЕ: Параформальдегид в «жабо» форме (сжатый небольшие гранулы) меньше, порошкообразные и поэтому может быть измерена вне капюшона. Однако вы все равно должны носить защитную маску от пыли (N95по крайней мере) во время обработки. EB Культура СМИ Для 500 мл EB Media: <table border="1"><tbody><tr><td> DMEM: </td><td> 404,5 мл </td></tr><tr><td> FBS: </td><td> 75,0 мл </td></tr><tr><td> L-Глютамин: </td><td> 5,0 мл </td></tr><tr><td> Пенициллина / стрептомицина: </td><td> 5,0 мл </td></tr><tr><td> Номера для незаменимых аминокислот: </td><td> 5,0 мл </td></tr><tr><td> Пирувата натрия: </td><td> 5,0 мл </td></tr><tr><td> β-меркаптоэтанол: </td><td> 500мкл </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 Video at JoVE.com

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

Приведем простой протокол для визуализации регионов запрограммированной клеточной смерти (PCD) у эмбрионов мышей и дифференциации эмбриональных стволовых (ЭС) клеточных культур с использованием высоко растворимых красителей называют LysoTracker.

Использование LysoTracker обнаружить запрограммированной смерти клеток в эмбрионы и дифференциации эмбриональных стволовых клеток

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

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