Optimization of calcium phosphate transfection method during delivering pMaxGFP into human hepatoma HepG2 cells

XU Hui; GONG Xia; PENG Jian-xiong

doi:10.3969/j.issn.1002-266X.2014.35.09

Abstract：Objective To optimize the application conditions of calcium phosphate transfection method when pMaxG-FP is delivered to human hepatoma HepG2 cells.Methods pMaxGFP was delivered to human hepatoma HepG2 cells of logarithmic growth phase by calcium phosphate transfection method.1 h before transfection, different serum concentrations of fresh medium which were null, 1%, 2%, 5%, 10%and 15%were changed to every culture wells.After 48 h, invert-ed fluorescence microscope was used to observe the expression of GFP to evaluate the transfection efficiency, while in situ try pan blue staining to detect cell viability.Thus, the optimal serum concentration was found.Then, under the condition of optimal serum concentration, the following experiment was divided into A, B, C, D, E five groups, namely adherent state, suspended 3 ×104 cells /well, 4 ×104 cells /well, 5 ×104 cells /well, 6 ×104 cells /well.After 48 h, inverted fluorescence microscope was used to observe the expression of GFP to evaluate the transfection efficiency, while in situ try pan blue staining to detect cell viability.Then the best state of cell and its density were optimized in the same way.Results After 48 h, there were a great number of dead cells in the serum-free group, while a little in the other group.In the 1%,2%, 5%, 10%, 15% serum concentration group, transfection efficiency was 30.92%±1.29%, 30.10%±1.05%, 21.27%±0.63%, 19.10%±0.51%, 10.44%±1.42%, respectively.Transfection efficiency was significantly higher than the other groups when the serum concentration was 1%and 2%, P<0.05;in the 1%, 2%, 5%, 10%, 15%serum concentration group, cell viability was 76.01%±0.94%, 89.68%±0.68%, 92.38%±1.02%, 93.36%±1.35%, 93.49%±0.91%, respectively.Cell viability in the 1%serum concentration group was significantly higher than the 2%. Under the condition of 2%serum concentration, transfection efficiency of A, B, C, D, E group was 30.74%±0.50%, 6.38%±0.28%, 31.41%±0.42%, 30.94%±0.47%, 1.41%±0.27%, respectively.A, C and D groups were sig-nificantly higher than B and E groups, P<0.05;cell viability of A, B, C, D, E group was 91.76% ±0.99%, 88.40%±1.29%, 88.38% ±1.08%, 87.48%±0.78%, 64.45% ±0.55%, respectively.A, B, C and D groups were sig-nificantly higher than E group.Conclusion The application conditions of calcium phosphate transfection method when pMaxGFP is delivered to human hepatoma HepG2 cells are 2%serum concentration and 4 ×104 cells/well or 5 ×104 cells/well when suspending transfection.