Alternatively, we suggest using this opportunity to take a little break from work and read some of the interesting articles below. This protocol describes a general method for transfecting mammalian cells using linear polyethylenimine. Hydroxychloroquine side effects ocular Plaquenil dose hydroxychloroquine Chloroquine autophagy lc3 Plaquenil makes swelling go down The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery. Skip to main content. Thank you for visiting. Transfection efficiency was affected by the concentration of lysosomotropic agents. The marked transfection enhancement was observed when the amount of sucrose increased from 5 to 500 mM and the optimum transfection efficiency was found at 500 mM in all cell lines tested Fig. 2 a–c. Chloroquine treatment of rodent cells during the first hours of polyoma DNA transfection increase the fraction of cells expressing viral functions. The effect has been observed after DNA absorption using both the DEAE-dextran and calcium phosphate coprecipitation. We specifically use this protocol with Lenti-X 293T cells, a cell line optimized for production of lentiviral vectors. Transfections allow for transient expression of a gene of interest in a target cell line and can be useful for short term studies of protein function. Chloroquine transfection lipofectamine Chloroquine use in transfection. - Tissue and Cell Culture, Enhanced plasmid DNA transfection with lysosomotropic agents. Chloroquine synthesis slideshare Add 22 l of 25mM chloroquine to each 15cm dish for 10cm plate 7.8 l of chloroquinei.e. final concentration = 25 M. The cells should be transfected within 5-10’ of adding the chloroquine. 7. For 15cm plates, add 1.69ml of the transfection buffer to the DNA-CaCl2 solution by “bubbling”. Transient Transfection of 293T cells. High efficiency polyoma DNA transfection of chloroquine.. Lipofectamine 2000 Transfection Reagent. We evaluated the transfection efficiency of Lipofectamine/pRSVβgal lipoplexes into HEK293 and C-33 A cells, alone or in the presence of increasing concentrations of a membrane fusion inducer chlorpromazine or procainamide plus the lysosomotropic agent chloroquine. Mar 03, 2014 The plasmids GFP-LC3 or small interfering RNA siRNA; Atg5 and Atg7 were transiently transfected into cells with Lipofectamine 2000 transfection or RNAi MAX reagent according to the manufacturer’s instructions Invitrogen. After 24 hours, the cells were treated with 5-FU or CQ and subjected to fluorescent analysis or Western blotting assay. Transfection efficiency was affected by the concentration of lysosomotropic agents. The marked transfection enhancement was observed when the amount of sucrose increased from 5 to 500 mM and the optimum transfection efficiency was found at 500 mM in all cell lines tested Fig. 2 a–c. Transgene expression was found to be very low when the cells were transfected with plasmid alone.