准备(参见解决方案在协议结束部分解决方案的组成) 准备培养皿。涂层玻璃Mattek玻璃底培养皿与聚- L -赖氨酸(四硼酸钠)。加入足够的完全覆盖的玻璃和离开> 1小时(可以过夜)。彻底清除聚- L -赖氨酸,在人工海水冲洗(ASW)的4-5倍。删除最后一次冲洗后,加2毫升50%L15(补充盐和含L -谷氨酰胺在终浓度为2mm)/ 50%血淋巴。这种培养基中必须至少一个小时,在盘前电镀细胞,…
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.