Summary

Анализ Магистральные нейронной клеточной миграции Crest с использованием модифицированного Zigmond пробирной палаты

Published: January 19, 2012
doi:

Summary

Подход к анализу миграции эксплантированных клеток (ствол клетки нервного гребня) описывается. Этот метод является недорогим, нежный, и способны различать хемотаксис с обеих хемокинез и других воздействий на миграционный полярности, таких как полученные из межклеточных взаимодействий в рамках первичного ствола нейронной клеточной культуре гребня.

Abstract

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9.

Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions.

Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

Protocol

1. День 1: Изоляция магистральных труб для нейронных ночной культуры на покровных Инкубируйте куриных яиц в течение 56 ч при 38 ° C. Удалите яйца от инкубации, мягко распылять их с 70% этанола, а затем дайте им высохнуть. Разбейте яйца в открытой УФ-стерилизованные стеклянные панели зада…

Discussion

Проведение исследования хемотаксиса на магистральных НКС оказалось сложным для ряда причин. Магистральные НКС представляют собой гетерогенную популяцию стволовых клеток, что будет отличать, если культурный долгосрочные, поэтому ствол НКС должно быть получено от первичного эксплант…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Мы даем особую благодарность Lino Ким, Стив Гусман и Ujit Satyarthi для оказания технической помощи в ходе разработки этого метода. Мирон Hawthorne, Ричард Spengel, и Роберто Рохас обрабатывается камеры, используемые здесь, и при условии, столь необходимую техническую помощь. Примечательно, что Роберто Рохас производится Рисунок 4. Мы также благодарны за ценные советы Скотт Фрейзер до развития выше анализ хемотаксиса. Эта работа была частично поддержана NIH-МУРЗ SCORE-5S06GM048680-13, Медб и награды от ХСС, Northridge Graduate Program Support Диссертация на CW.

Materials

Name of the reagent Company Catalogue number Comments (optional)
DMEM Omega Scientific DM-22  
Penicillin Streptomycin Solution Omega Scientific PS-20 100X Stock Concentration
L-Glutamine Omega Scientific GS-60 100X Stock Concentration
Fetal Bovine Serum Omega Scientific FB-11 Lot# 105247 (or another that is comparable)
Modified Zigmond chamber Home made N/A Reservoir volume: ~ 160 μl ea; for additional specifications, see Fig. 4 and the supplemental fabrication protocol
Cell culture dish Denville T6040 40 x 10 mm
Fibronectin BD 354008 10X Stock prepped by diluting 1 mg FN in 1 ml H2O and 9 ml DMEM
Coverslips Fisher 12-548-B Precleaned; 22 x 22 mm
L15 medium Thermo Scientific SH30525.02  
Petroleum Jelly Comforts 011110794642 100%
Centrifuge tube Biologix 10-9152 15 ml
Dispase Cell Systems 4Z0-850 10X Stock Concentration
Syringe BD 309602 1 ml
Needle BD 305127 25 G x 1.5 in.
Alexa Fluor 488-IgM Invitrogen A21042 Stock is 2 mg/ml; 7 moles dye/mole IgM
Dissecting Forceps FST Misc. Dumont #5 or 55; straight tipped; stainless steel or titanium
Tungsten Needle N/A N/A Home made; placed in a pin holder
Blunt Forceps Tiemann 160-18 Used for transferring embryos to Ringer’s from egg yolk

Supplemental Protocol: Fabrication of a Modified Zigmond Chamber

Please refer to Figure 4 as a reference for the protocol below:

  1. Purchase a sheet of 3/16″ thick polished acrylic (4.45 mm actual thickness).
  2. Using a table saw, cut chamber blanks oversized to the rough dimensions of 33.25 mm x 64.57 mm. This allows 3.175 mm extra material for machining.
  3. Set the chamber blank on a vise. With a milling machine and a 6.35 mm (1/4″) end mill bit, finish machining the sides of the chamber to their exact dimensions: 30.07 mm x 61.39 mm.
  4. Position the chamber blank on the milling machine and locate the center of the blank along both the x and y axes with an edge finder; then zero the center location.
  5. Acquire the chamber height (z-axis) by touching the end mill bit to the top surface and zero the height.
  6. Using a 3.91 mm (0.154″) end mill bit, offset the bit 3.03 mm along the x-axis (positive direction) for the first reservoir. Begin machining into the chamber to a depth of 2.84 mm while moving along the y-axis (positive direction) to 7.62 mm (0.300″) and then traverse to 7.62 mm (0.300″) in the opposite (negative) direction to a complete reservoir length of 15.24 mm (0.600″). Offset the bit to 3.03 mm (0.119″) along the x-axis (negative direction) and repeat the same process for the second reservoir.
  7. Position the chamber on its edge and drill a hole using a 1.09 mm (0.043 in.) drill bit on the end of each reservoir (4 total) that connects the end of the reservoir to the side of the chamber for loading medium during experimentation.
  8. Soak the chamber well in warm soapy water to help remove any chemical contaminants.
  9. Soak and rinse the chamber well in double-distilled water to remove any soap. The chambers are now ready to use as described above.

References

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  2. Baker, C. V. . Neural Crest and Cranial Ectodermal Placodes. , (2005).
  3. Gammill, L. S., Roffers-Agarwal, J. Division of labor during trunk neural crest development. Dev. Biol. 344, 555-565 (2010).
  4. Kulesa, P. M., Gammill, L. S. Neural crest migration: patterns, phases and signals. Dev. Biol. 344, 566-568 (2010).
  5. Wang, H. U., Anderson, D. J. Eph family transmembrane ligands can mediate repulsive guidance of trunk neural crest migration and motor axon outgrowth. Neuron. 18, 383-396 (1997).
  6. Krull, C. E. Interactions of Eph-related receptors and ligands confer rostrocaudal pattern to trunk neural crest migration. Curr. Biol. 7, 571-580 (1997).
  7. Gammill, L. S., Gonzalez, C., Gu, C., Bronner-Fraser, M. Guidance of trunk neural crest migration requires neuropilin 2/semaphorin 3F signaling. Development, Cambridge, England. , 133-199 (2006).
  8. De Bellard, M. E., Rao, Y., Bronner-Fraser, M. Dual function of Slit2 in repulsion and enhanced migration of trunk, but not vagal, neural crest cells. The Journal of cell biology. 162, 269-279 (2003).
  9. Kasemeier-Kulesa, J. C., McLennan, R., Romine, M. H., Kulesa, P. M., Lefcort, F. CXCR4 controls ventral migration of sympathetic precursor cells. J. Neurosci. 30, 13078-13088 (2010).
  10. Zigmond, S. H. Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors. The Journal of Cell Biology. 75, 606-616 (1977).
  11. Hamburger, V., Hamilton, H. L. A series of normal stages in the development of the chicken embryo. J. Morph. 88, 49-52 (1951).
  12. Boyden, S. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. The Journal of Experimental Medicine. 115, 453-466 (1962).
  13. Davis, E. M., Trinkaus, J. P. Significance of cell-to cell contacts for the directional movement of neural crest cells within a hydrated collagen lattice. Journal of Embryology and Experimental Morphology. 63, 29-51 (1981).
  14. Zicha, D., Dunn, G. A., Brown, A. F. A new direct-viewing chemotaxis chamber. Journal of Cell Science. 99, 769-775 (1991).

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Cite This Article
Walheim, C. C., Zanin, J. P., de Bellard, M. E. Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay. J. Vis. Exp. (59), e3330, doi:10.3791/3330 (2012).

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