L929是小鼠成纤维细胞瘤细胞株，经常被用来检测TNF-alpha及TNF-beta。对TNF的处理经常会引发细胞凋亡及死亡，因此L929细胞经常被用作免疫分析。但是，转染L929细胞非常难，尤其使用基于脂质体技术的转染试剂，我们使用GenJet VerⅡ及PolyJet转染L929细胞获得了75%的转染效率，简单的实验步骤如下。
1、转染时确保L929细胞达到80%的融合度，细胞必须健康并且传代不能超过9代。
2、对于6孔板，用不含血清的DMEM分别稀释1.0µg，DNA及3.0µl，Genjet VerⅡ或PolyJet转染试剂。将稀释好的转染试剂加入DNA中，室温下放置15分钟以形成转染复合物，其它规格的细胞培养器皿，可以根据表面积适当调整DNA含量。
3、将转染复合物直接加入L929细胞中；6孔板，每孔含1.0ml培养基，在血清/抗生素存在下,转染进行。
4、转染后的24~48小时，监测转染基因的表达情况。
Brief procedures for transfecting L929 cell.
L929 is a murine aneuploid fibrosarcoma cell line which is often used to assay TNF-alpha and TNF-beta. Treatment with TNF initiates apoptosis and subsequent cell death, therefore L929 cell is often used for immunological assays. However L929 cell is hard to transfect, especially resistant to liposome based transfection method. We have been using GenJet Ver. II and PolyJet to transfect L929 cell and got up to 75% efficiency. The brief procedures are described below for transfecting L929 cells with GenJet and PolyJet.
1. Grow L929 cell to ~80% confluency at the day of transfection. L929 cell must be healthy and less than 9 passages for maximum efficiency.
2. For 6-well plate, dilute 1.0 µg of DNA and 3.0 µl of GenJet Ver. II or PolyJet reagents per well with serum free DMEM respectively. Add diluted reagent to diluted DNA and let transfection complex formed at RT for 15 minutes. For other format of cell culture formats, scale down or up per the surface area of culture dish.
3. Add transfection complex to L929 cell directly. Transfection is conducted in presence of serum/antibiotics with transfection volume of 1.0 ml per well of 6-well plate.
4. Check transgene expression 24~48 hours post transfection.
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