小中大回复 #311 yysr238 的帖子
2:灌注液的配置:
Perfusion Solution 1(g/L):
NaCL 8.000
NaH2PO4•2H2O 0.078
KCL 0.400
Na2HPO4•12H2O 0.151
NaHCO3 0.350
EDTA 0.190
HEPES 2.380
Glucose 0.900
磁力搅拌器使固体成分充分溶解,用1M HCL或1M NaOH调定pH 7.2~7.4(使用pH计), 0.45及0.22双层滤膜负压过滤除菌,4℃保存,使用时液体温度维持在37℃(应用水浴箱)。
Perfusion Solution 2(g/L):
NaCL 8.000
KCL 0.400
CaCL2 0.560
NaH2PO4•2H2O 0.078
Na2HPO4•12H2O 0.151
HEPES 2.380
NaHCO3 0.350
Collagenase Ⅳ 0.500
搅拌器使固体成分充分溶解,4℃冰箱过夜,以后同上。
4:灌注步骤:
4.1 37℃Perfusion Solution 1沿门静脉插管灌注肝脏,流速20-30ml /min,灌洗10min左右,至肝脏呈现灰白色为止
4.2 Hanks液80ml 短时间灌注,约2min,冲出EDTA
4.3 37℃Perfusion Solution 2 沿门静脉插管灌注肝脏,流速20mL/min,灌洗10min左右,循环使用
4.4 肝脏变软,塌陷后用无菌镊钝性分离肝细胞,去除肝包膜及血管等结缔组织,将含有胶原酶的肝细胞悬液放入250ml无菌烧杯中(杯口用无菌锡纸包裹),37℃水浴5min
4.5加入无指示剂的DMEM基培,双层无菌纱布过滤
4.6将滤液放入50ml离心管中,应用无指示剂的DMEM基培,4℃、50g、3min洗涤肝细胞3次
5:获得的细胞经过以下的纯化,应用特定的培养基培养。
提供几篇文章
1. Klaunig, J. E., Goldblatt, P. J., Hinton, D. E., Lipsky, M. M., Chacko, J., and Trump, B. F. (1981). Mouse liver cell culture 1. hepatocyte isolation. IN VITRO Vol.17.No.10 October, 913-925.
2. Kreamer, B. L., Staecker, J. L., Sawada, N., Sattler, G. L., Hsia, M. T. S., and Pitot, H. C. (1986) Use of low-speed ,iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations. IN VITRO CELLULAR & DEVELOPMENTAL BIO. Volume 22,Number 4,April 201-211.
3. Sambrook, J., Fritsch, E. F., Maniatis, T., (1989) Lysis of cultured mammalian cells. Molecular Cloning. 18.34