विच्छेदन और neurite परिणाम Assays के लिए चिकी Statoacoustic नाड़ीग्रन्थि और कोलेजन जैल में स्पाइनल कॉर्ड Explants की संस्कृति

Summary

हम प्रदर्शन कैसे टुकड़े करना और संस्कृति लड़की E4 statoacoustic नाड़ीग्रन्थि और E6 रीढ़ की हड्डी explants. Explants 24 घंटे के लिए 3 डी कोलेजन जैल में सीरम मुक्त शर्तों के तहत सुसंस्कृत हैं. Neurite जवाबदेही कारक पूरक मध्यम विकास के साथ और प्रोटीन में लिपटे मोतियों के साथ परीक्षण किया है.

Abstract

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues¹ and survival factors² located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons³. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons⁴. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro^5,6.

Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth.

SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol⁷) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival⁸.

Protocol

व्यंजनों चिकी Ringers 7.2 छ NaCl 0.23 छ CaCl 2 + 2 हे 2H 0.37 छ KCl .115 छ ना दो चार HPO 900 मिलीलीटर जल (टिशू कल्चर ग्रेड) नोट: 7.4 पीएच …

Discussion

हम एक विधि टुकड़े करना और संस्कृति E4 शिथिलता और E6 रीढ़ की हड्डी explants, लड़की से सीरम मुक्त शर्तों के तहत, वर्तमान. यह प्रक्रिया वर्तमान में हमारी प्रयोगशाला में प्रयोग किया जाता है शिथिलता न्यूरॉन अस्तित्…

Materials

Reagent	Company	Catalogue number	Comment
Equipment
Dissection microscope			We use a Leica M80 stereomicroscope with brightfield base illumination
Slide warmer	C.S. & E.		Collagen polymerization
Chicken egg incubator
37°C/5% CO2 humidified cell culture incubator
Dissection Materials
Sylgard© coated petri dish
24-well cell culture plate	Corning	3526
#5 Dumont forceps	Fine Science Tools	11252-30
#55 Dumont forceps	Fine Science Tools	11295-51
Stainless steel dissecting pins	Fine Science Tools	26002-10	Embryo pinning, fine dissection
Moria perforated spoon	Fine Science Tools	10370-17	Embryo harvest/transfer
200 µl wide mouth pipet tips	Dot Scientific Inc	118-96R	Explant transfer
E4 and E6 chicken eggs
Chick Ringer’s solution			Embryo harvest
HBSS	Sigma	H8264	SAG dissection
L15 medium	Sigma	L5520	Spinal cord dissection medium
Fetal calf serum	Atlanta Biologicals	S11150	Spinal cord dissection medium
Vannas Scissors	World Precision Instruments	501778	Spinal cord dissection
Collagen preparation
Rat-tail collagen Type I	BD Biosciences	354249	Explant culture
10X PBS (Sterile)			To neutralize collagen
1N NaOH (Sterile)			To neutralize collagen
Distilled water (Sterile)			To neutralize collagen
pH indicator papers (6.0-8.1)	Whatman	2629-990	To check collagen pH
15 ml conical tubes	Dot Scientific Inc	818-PG
500 µl wide mouth pipet tips	Dot Scientific Inc	119-R100	To pipet collagen
Explant culture
DMEM/F12 medium	Sigma	D8437	Explant culture medium
ITS+1	Sigma	I2521	Medium supplement
Penicillin-Streptomycin	Invitrogen	15140-122	Medium supplement
CNTF (rat)	Sigma	C3835	Medium supplement
NT-3 (human)	Sigma	N1905	Medium supplement
Purified mouse Wnt5a	R&D Systems	645-WN-010
Affi-gel Blue gel beads	Bio-Rad	153-7302
Immunohistochemistry
Paraformaldehyde	Sigma	P6148	Fixation
PBS			Rinses
Triton X-100	Sigma	T9284	Blocking solution
Sodium azide	Sigma	S8032	Blocking solution
Calf serum	Invitrogen	16170-078	Blocking solution
Mouse monoclonal IgG2b anti-β Tubulin 1+II antibody	Sigma	T8535	Labels cell bodies and neurites
Alexa fluor 488 goat anti –mouse IgG2b antibody	Invitrogen	A21141	Detects β Tubulin antibody
Teflon micro spatula	VWR	57949-033	Release collagen gels from well