Оптимизирована процедура для флуоресцентной активированного сортировки клеток (FACS) Выделение вегетативной нервной прародителей из висцеральных органов плода мышей
Summary
Оптимизированные процедуры для очищения нервного гребня, полученные от предшественников нейронов фетальных тканей мыши описано. Этот метод использует выражение из флуоресцентного аллелей репортер, чтобы изолировать отдельные группы по флуоресценции активированной сортировки клеток (FACS). Этот метод может применяться для изоляции нейронов субпопуляции на протяжении всего развития или из взрослых тканей.
Abstract
During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the intestine and lower urinary tract. Both during development and in mature tissues these cells are often widely dispersed throughout tissues so that isolation of discrete populations using methods like laser capture micro-dissection is difficult. They can however be directly visualized by expression of fluorescent reporters driven from regulatory regions of neuron-specific genes like Tyrosine hydroxylase (TH). We describe a method optimized for high yields of viable TH+ neuronal progenitors from fetal mouse visceral tissues, including intestine and lower urogenital tract (LUT), based on dissociation and fluorescence-activated cell sorting (FACS).
The Th gene encodes the rate-limiting enzyme for production of catecholamines. Enteric neuronal progenitors begin to express TH during their migration in the fetal intestine1 and TH is also present in a subset of adult pelvic ganglia neurons2-4 . The first appearance of this lineage and the distribution of these neurons in other aspects of the LUT, and their isolation has not been described. Neuronal progenitors expressing TH can be readily visualized by expression of EGFP in mice carrying the transgene construct Tg(Th-EGFP)DJ76Gsat/Mmnc1. We imaged expression of this transgene in fetal mice to document the distribution of TH+ cells in the developing LUT at 15.5 days post coitus (dpc), designating the morning of plug detection as 0.5 dpc, and observed that a subset of neuronal progenitors in the coalescing pelvic ganglia express EGFP.
To isolate LUT TH+ neuronal progenitors, we optimized methods that were initially used to purify neural crest stem cells from fetal mouse intestine2-6. Prior efforts to isolate NC-derived populations relied upon digestion with a cocktail of collagenase and trypsin to obtain cell suspensions for flow cytometry. In our hands these methods produced cell suspensions from the LUT with relatively low viability. Given the already low incidence of neuronal progenitors in fetal LUT tissues, we set out to optimize dissociation methods such that cell survival in the final dissociates would be increased. We determined that gentle dissociation in Accumax (Innovative Cell Technologies, Inc), manual filtering, and flow sorting at low pressures allowed us to achieve consistently greater survival (>70% of total cells) with subsequent yields of neuronal progenitors sufficient for downstream analysis. The method we describe can be broadly applied to isolate a variety of neuronal populations from either fetal or adult murine tissues.
Protocol
Discussion
Мышь репортер линии, экспрессирующие флуоресцентные журналистам становятся доступными через многочисленные усилия в мышиной генетики сообщество 1,8,9. В результате метод диссоциации показано здесь может широко применяться для изоляции дискретных нейронов подтипы на основе мед?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Авторы хотели бы поблагодарить Екатерину за Alford предложения по клеточная диссоциация методы и Кевин Уэллер, Дэвид Флаэрти и Бретани Мэтлок за поддержку в проточной цитометрии общих ресурсов в Университете Вандербильта медицинский центр и Мелисса А. Musser художественного помощь с иллюстрациями. Мы благодарим доктора. Джек Мошер и Шон Моррисон совета по реализации изоляции нейронов прародителей. VMC проточной цитометрии общих ресурсов поддерживается Вандербильта Ingram Cancer Center (P30 CA68485) и пищеварительной Вандербильта болезни Научно-исследовательский центр (P30 DK058404). Эта работа была поддержана финансирование от американского Национального института здравоохранения грантов DK064251, DK086594 и DK070219.
Materials
| Reagent Name | Vendor | Catalog number | Comments |
| Accumax | Sigma(mfr: Innovative Cell Technologies) | A7089-100ML | Store frozen in 1 ml aliquots |
| DNase I | Sigma | D-4527 | Stored frozen at -20 °C 5 mg/ml in 1xHBSS, (Used in Quench, Quench 1:5) |
| 10X PBS pH 7.4 | Gibco | 70011-044 | Make up to 1x with tissue culture grade water then sterile filter |
| 10X HBSS w/o Ca or Mg | Gibco | 14185-052 | Make up to 1x with tissue culture grade water then sterile filter |
| Leibovitz’s L-15 medium | Gibco | 21083027 | |
| Penicillin /Streptomycin 100X | Gibco | 15140-133 | Store aliquoted at -20 °C |
| BSA | Sigma | A3912-100G | Store aliquoted at -20 °C, 100 mg/ml in water |
| Biowhittaker 1M HEPES in 0.85% NaCl | Lonza | 17-737E | |
| 38 μm NITEX Nylon Mesh Membrane | Sefar America | 3-38/22 | Cut into ~3 cm squares. UV treat overnight to sterilize in the tissue culture hood. |
| 7-AAD | Invitrogen | A1310 | 1 mg/ml |
| TRIzol LS | Invitrogen | 10296-028 | |
| 5 ml polystyrene tubes | Falcon | 352058 | |
| 15 ml conical tubes | Corning | 430790 | |
| Fine Dissecting Forceps | Fine Science Instruments | 11251-30 | Dumont#5 forcep, Dumoxel, standard tip 0.1×0.06mm |
| Dissecting Spoon | Fine Science Instruments | 10370-18 |
Riferimenti
- Gong, S. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature. 425, 917-925 (2003).
- Morrison, S. J., White, P. M., Zock, C., Anderson, D. J. Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells. Cell. 96, 737-749 (1999).
- Kruger, G. M. Neural crest stem cells persist in the adult gut but undergo changes in self-renewal, neuronal subtype potential, and factor responsiveness. Neuron. 35, 657-669 (2002).
- Bixby, S., Kruger, G. M., Mosher, J. T., Joseph, N. M., Morrison, S. J. Cell-intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity. Neuron. 35, 643-656 (2002).
- Walters, L. C., Cantrell, V. A., Weller, K. P., Mosher, J. T., Southard-Smith, E. M. Genetic background impacts developmental potential of enteric neural crest-derived progenitors in the Sox10Dom model of Hirschsprung disease. Human Molecular Genetics. , (2010).
- Corpening, J. C. Isolation and live imaging of enteric progenitors based on Sox10-Histone2BVenus transgene expression. Genesis. 49, 599-618 (2011).
- Morrison, S. J. . Isolation of fetal rat neural crest stem cells (NCSC) from gut and sciatic nerve. , (2012).
- Skarnes, W. C. A conditional knockout resource for the genome-wide study of mouse gene function. Nature. 474, 337-342 (2011).
- Harding, S. D. The GUDMAP database–an online resource for genitourinary research. Development. 138, 2845-2853 (2011).
- Joseph, N. M. Enteric glia are multipotent in culture but primarily form glia in the adult rodent gut. J. Clin. Invest. 121, 3398-3411 (2011).
- Newgreen, D. F., Murphy, M. Neural crest cell outgrowth cultures and the analysis of cell migration. Methods Mol. Biol. 137, 201-211 (2000).
