Optimizing condition for lipofectin-mediated NF-?B decoy oligodeoxynucleotides transfection into J774.1 cells
- VernacularTitle:阳离子脂质体介导NF-?B“decoy”ODNs转染巨噬细胞条件的优化
- Author:
Jian WANG
;
Jinbao LI
;
Xuyi REN
;
Xiaoming DENG
;
- Publication Type:Journal Article
- Keywords:
NF ?B decoy oligodeoxynucleotides;
lipofectin;
transfection;
macrophage
- From:
Academic Journal of Second Military Medical University
2000;0(08):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To search for the optimizing parameters and distribution pattern of NF ?B decoy oligodeoxynucleotides in transfecting J774.1 cells mediated by lipofectin. Methods: With the change of ODNs/lipofectin ratio and transfection time, the uptake rate and mean fluorescence intensity of NF ?B decoy oligodeoxynucleotides in J774.1 cells were measured by flow cytometry to evaluate transfection efficiencies. Intracellular distribution of NF ?B decoy oligodeoxynucleotides was determined with fluorescence microscopy. The lactate dehydrogenase (LDH) activity in the supernatant was assayed to assess the cytotoxicity. Results: The uptake of NF ?B decoy oligodeoxynucleotides into J774.1 cells was significantly improved by lipofectin. In 24 well culture plate, 1 ?g ODNs with 5?g lipofectin ( W/W =1∶5) resulted in the highest transfection efficiency and lower cytotoxicity. The NF ?B decoy oligodeoxynucleotides localized in the nucleus as well as in the cytoplasm following an incubation of 6 h with lipofectin. While NF ?B decoy oligodeoxynucleotides had faint fluorescences in cytoplasm in the absence of lipofectin. Conclusion: lipofectin can enhance the cellular uptake of NF ?B decoy oligodeoxynucleotides in J774.1 cells and alter intracellular distribution of NF ?B decoy oligodeoxynucleotides. Efficiency of transfection is the highest when the ratio of ODNs/lipofectin is 1∶5 for incubation of 6 h.
