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

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…

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.