活体成像用huh7-luc细胞株

Huh-7-luc 肝癌细胞

1.       细胞名称    Huh-7-luc

2.       组织  肝癌

3.       母细胞来源  ATCC

4.       转染方法与标记过程的描述：慢病毒转染筛选单克隆 

Construct Luciferase expressing vector pWPXL-Luc.

pGL3-control was first digested with XbaI and then blunted with Klenow. The resulted DNA was then digested with BglII. The DNA fragment (1902 bp) encoding luciferase was then ligated to BamHI/SmaI backbone of pWPXL.

Preparation of the lentivirus.

In the first day, 2.5×106 of 293T cells was plated in 10cm plate.

In the next day, 20μg of pWXL-Luc, 15 μg of psPAX2 and 6 μg of PMD2G was diluted into a tube containing 500 μl H2O，and then 500 μl 2xHBS was supplemented into the tube。50 μl of 2.5 M CaCl2 was added and mixed gently. The solution was incubated 20~25min at RT。After that, the solution was added into the medium。After 6 hr, the medium was replaced with fresh medium。

The fourth day, medium was collected at 3000 rpm, RT for 5 min. The virus was purified by passed through with 0.45 μm membrane filters.

Establishment of HuH-7-Luc cell line.

pWXL-Luc Virus were used to infect the huh-7 cells at MOI=1000 in the presence of 4μg/ml polybrene. After 3 days, cells were counted, diluted and plated into 96-well plate by about one cell per well. After 2~3 weeks, the expression of the luciferase in the cell clones was examined. Cell clone with high expression of luciferase was selected and stored.

5.       培养条件 ：置于37℃、5％CO2培养箱中，用含10％胎牛血清（FBS;    GIBCO）和1％双抗（100 U/ ml青霉素和100 μg/ ml链霉素，GIBCO）的DMEM（高糖），培养三天（密度大概80%）传代。

【注意事项】本细胞标记前与标记后在形态上略有差别，属于正常现象，如下图所示。左图是标记前形态，右图是标记后形态。

6.       细胞传代所需时间：3天/代 

7.       选择压力：无

8.       体外实验数据：细胞读数为1194 RLU/cell。  

9.       体内实验 ：肝原位接种细胞3×10的6，两周后活体荧光成像。