Summary

Оптимизирована процедура для флуоресцентной активированного сортировки клеток (FACS) Выделение вегетативной нервной прародителей из висцеральных органов плода мышей

Published: August 17, 2012
doi:

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

1. Подготовка Media (все шаги сделал в ткани капот культуры) Смешайте следующие: 44 мл L-15 средняя, ​​0,5 мл 100X пенициллина / стрептомицина (P / S), 0,5 мл 100 мг / мл бычьего сывороточного альбумина (БСА), 0,5 мл 1М HEPES, 5 мл культуры ткани класса воде. Будьте уверены, чтобы смешивать BSA и P / S тщательно…

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?…

Disclosures

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  

References

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Cite This Article
Buehler, D. P., Wiese, C. B., Skelton, S. B., Southard-Smith, E. M. An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice. J. Vis. Exp. (66), e4188, doi:10.3791/4188 (2012).

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