の初代培養<em>アメフラシ</em>感覚運動ニューロンは、シナプス形成とシナプス可塑性を研究するためのモデルの準備をしなくては<em> in vitroで</em>。このビデオでは、より感覚および運動ニューロンの同定とマイクロダイセクションを示しています<em>アメフラシ</em>節と同様に文化の中で感覚運動ニューロンを確立し、維持するための方法。
準備(ソリューションの構成のためのプロトコルの終了時にソリューションを参照してください) 培養皿を準備します。ポリ- L -リジン(ホウ酸ナトリウムで作られた)とマテックガラス底培養皿のよくコートガラス。完全によくガラスをカバーし、> 1時間(一晩放置することができます)に向けて出発するのに十分な追加。徹底的に人工海水(ASW)4-5回ですすぐことにより、ポリ…
0.35 M MgCl2, stored at room temperature, used for anesthesia.
Poly-L-lysine solution, Sigma: P-1524, MW >300,000, made in 0.1M Sodium Borate pH8.2 to 0.5mg/ml solution. Vortex well and filter sterilize through a 0.22 μm filter, store it at 4C°. Do not freeze-thaw.
L-15 Medium powder (Leibovitz) (Sigma: L4386) supplemented with the salts as below to make 1 liter
L-15 powder
13.8g
NaCl
15.4 g
D-Glucose
6.24 g
MgSO47H20
6.45g
KCl
350 mg
NaHCO3
170 mg
MgCl26H2O
5.49 g
CaCl22H2O
1.43g
HEPES
3.53g
Add ddH20 to 1 liter. The pH should be about 7.4-7.5, add 10ml of 100X Pen/Strep solution, and filter-sterilize through a 0.22 µm filter. Store at 4C0 for no longer than 1 month.
Protease digestion solution: 1% Protease IX (1unit/mg) is made in L15 (supplemented with salts as above) or in ASW immediately before use, filter-sterilized through a 0.22 µm Millipore. 5mls should be enough for the ganglia from two animals (make sure the ganglia are completely immersed in protease solution). Sigma has reported that they will discontinue selling Protease IX. A substitute protease is: Dispase II (Roche Applied Science catalog # 04942078001).
Culture medium. Immediately prior to preparing cultures, thaw a 10 ml aliquot of hemolymph and mix it with 10 mls of L15 (supplemented with salts as above) to make 20mls culture medium. Add 200 μl of 200mM L- Glutamine, mix well and use for preparing cultures. This medium should be prepared fresh each time cultures are made.
Artificial Seawater can be made from Instant Ocean (Aquarium Systems, Mentor, OH) or as follows: 450 mM NaCl, 10 mM KCl, 30 mM MgCl2(6H2O), 20 mM MgSO4, 10 mM CaCl2(2H2O), 10 mM HEPES, with pH adjusted to 7.4.